Therapeutic compositions

ABSTRACT

This application relates to therapeutic siRNA agents and methods of making and using the agents.

RELATED APPLICATIONS

The present application is a continuation of U.S. application Ser. No. 14/943,612, filed Nov. 17, 2015, which is a continuation of U.S. application Ser. No. 14/282,769, filed May 20, 2014, which is a continuation of U.S. application Ser. No. 13/626,196, filed Sep. 25, 2012, which is a continuation of U.S. application Ser. No. 12/721,413, filed Mar. 10, 2010, which is a continuation of U.S. application Ser. No. 10/548,611, filed Aug. 22, 2006, which is the National Stage of International Application No. PCT/US2004/007070, filed Mar. 8, 2004, which claims the benefit of Application No. 60/452,682, filed Mar. 7, 2003; Application No. 60/462,894, filed Apr. 14, 2003; and Application No. 60/465,665, filed Apr. 25, 2003; Application No. 60/463,772, filed Apr. 17, 2003; Application No. 60/465,802, filed Apr. 25, 2003; Application No. 60/493,986, filed Aug. 8, 2003; Application No. 60/494,597, filed Aug. 11, 2003; Application No. 60/506,341, filed Sep. 26, 2003; Application No. 60/518,453, filed Nov. 7, 2003; Application No. 60/454,265, filed Mar. 12, 2003; Application No. 60/454,962, filed Mar. 13, 2003; Application No. 60/455,050, filed Mar. 13, 2003; Application No. 60/469,612, filed May 9, 2003; Application No. 60/510,246, filed Oct. 9, 2003; Application No. 60/510,318, filed Oct. 10, 2003. The contents of the above applications are hereby incorporated by reference in their entirety.

TECHNICAL FIELD

The invention relates to RNAi and related methods, e.g., methods of making and using iRNA agents.

BACKGROUND

RNA interference or “RNAi” is a term initially coined by Fire and co-workers to describe the observation that double-stranded RNA (dsRNA) can block gene expression when it is introduced into worms (Fire et al. (1998) Nature 391, 806-811). Short dsRNA directs gene-specific, post-transcriptional silencing in many organisms, including vertebrates, and has provided a new tool for studying gene function. RNAi may involve mRNA degradation.

SUMMARY

A number of advances related to the application of RNAi to the treatment of subjects are disclosed herein. For example, the invention features iRNA agents targeted to specific genes; palindromic iRNA agents; iRNA agents having non canonical monomer pairings; iRNA agents having particular structures or architectures e.g., the Z-X-Y or asymmetrical iRNA agents described herein; drug delivery conjugates for the delivery of iRNA agents; amphipathic substances for the delivery of iRNA agents, as well as iRNA agents having chemical modifications for optimizing a property of the iRNA agent. The invention features each of these advances broadly as well as in combinations. For example, an iRNA agent targeted to a specific gene can also include one or more of a palindrome, non canonical, Z-X-Y, or asymmetric structure. Other nonlimiting examples of combinations include an asymmetric structure combined with a chemical modification, or formulations or methods or routes of delivery combined with, e.g., chemical modifications or architectures described herein. The iRNA agents of the invention can include any one of these advances, or pairwise or higher order combinations of the separate advances.

In one aspect, the invention features iRNA agents that can target more than one RNA region, and methods of using and making the iRNA agents.

In another aspect, an iRNA agent includes a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region.

In one embodiment, the first and second sequences of the iRNA agent are on different RNA strands, and the mismatch between the first and second sequences is less than 50%, 40%, 30%, 20%, 10%, 5%, or 1%.

In another embodiment, the first and second sequences of the iRNA agent are on the same RNA strand, and in a related embodiment more than 50%, 60%, 70%, 80%, 90%, 95%, or 1% of the iRNA agent is in bimolecular form.

In another embodiment, the first and second sequences of the iRNA agent are fully complementary to each other.

In one embodiment, the first target RNA region is encoded by a first gene and the second target RNA region is encoded by a second gene, and in another embodiment, the first and second target RNA regions are different regions of an RNA from a single gene. In another embodiment, the first and second sequences differ by at least 1 and no more than 6 nucleotides.

In certain embodiments, the first and second target RNA regions are on transcripts encoded by first and second sequence variants, e.g., first and second alleles, of a gene. The sequence variants can be mutations, or polymorphisms, for example.

In certain embodiments, the first target RNA region includes a nucleotide substitution, insertion, or deletion relative to the second target RNA region.

In other embodiments, the second target RNA region is a mutant or variant of the first target RNA region.

In certain embodiments, the first and second target RNA regions comprise viral, e.g., HCV, or human RNA regions. The first and second target RNA regions can also be on variant transcripts of an oncogene or include different mutations of a tumor suppressor gene transcript. In one embodiment, the oncogene, or tumor suppressor gene is expressed in the liver. In addition, the first and second target RNA regions correspond to hot-spots for genetic variation.

In another aspect, the invention features a mixture of varied iRNA agent molecules, including one iRNA agent that includes a first sequence and a second sequence sufficiently complementary to each other to hybridize, and where the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region. The mixture also includes at least one additional iRNA agent variety that includes a third sequence and a fourth sequence sufficiently complementary to each other to hybridize, and where the third sequence is complementary to a third target RNA region and the fourth sequence is complementary to a fourth target RNA region. In addition, the first or second sequence is sufficiently complementary to the third or fourth sequence to be capable of hybridizing to each other. In one embodiment, at least one, two, three or all four of the target RNA regions are expressed in the liver. Exemplary RNAs are transcribed from the apoB-100 gene, glucose-6-phosphatase gene, beta catenin gene, or an HCV gene.

In certain embodiments, the first and second sequences are on the same or different RNA strands, and the third and fourth sequences are on same or different RNA strands.

In one embodiment, the mixture further includes a third iRNA agent that is composed of the first or second sequence and the third or fourth sequence.

In one embodiment, the first sequence is identical to at least one of the second, third and fourth sequences, and in another embodiment, the first region differs by at least 1 but no more than 6 nucleotides from at least one of the second, third and fourth regions.

In certain embodiments, the first target RNA region comprises a nucleotide substitution, insertion, or deletion relative to the second, third or fourth target RNA region.

The target RNA regions can be variant sequences of a viral or human RNA, and in certain embodiments, at least two of the target RNA regions can be on variant transcripts of an oncogene or tumor suppressor gene. In one embodiment, the oncogene or tumor suppressor gene is expressed in the liver.

In certain embodiments, at least two of the target RNA regions correspond to hot-spots for genetic variation.

In one embodiment, the iRNA agents of the invention are formulated for pharmaceutical use. In one aspect, the invention provides a container (e.g., a vial, syringe, nebulizer, etc) to hold the iRNA agents described herein.

Another aspect of the invention features a method of making an iRNA agent. The method includes constructing an iRNA agent that has a first sequence complementary to a first target RNA region, and a second sequence complementary to a second target RNA region. The first and second target RNA regions have been identified as being sufficiently complementary to each other to be capable of hybridizing. In one embodiment, the first and second target RNA regions are on transcripts expressed in the liver.

In certain embodiments, the first and second target RNA regions can correspond to two different regions encoded by one gene, or to regions encoded by two different genes.

Another aspect of the invention features a method of making an iRNA agent composition. The method includes obtaining or providing information about a region of an RNA of a target gene (e.g., a viral or human gene, or an oncogene or tumor suppressor, e.g., p53), where the region has high variability or mutational frequency (e.g., in humans). In addition, information about a plurality of RNA targets within the region is obtained or provided, where each RNA target corresponds to a different variant or mutant of the gene (e.g., a region including the codon encoding p53 248Q and/or p53 249S). The iRNA agent is constructed such that a first sequence is complementary to a first of the plurality of variant RNA targets (e.g., encoding 249Q) and a second sequence is complementary to a second of the plurality of variant RNA targets (e.g., encoding 249S). The first and second sequences are sufficiently complementary to hybridize. In certain embodiments, the target gene can be a viral or human gene expressed in the liver.

In one embodiment, sequence analysis, e.g., to identify common mutants in the target gene, is used to identify a region of the target gene that has high variability or mutational frequency. For example, sequence analysis can be used to identify regions of apoB-100 or beta catenin that have high variability or mutational frequency. In another embodiment, the region of the target gene having high variability or mutational frequency is identified by obtaining or providing genotype information about the target gene from a population. In another embodiment, the genotype information can be from a population suffering from a liver disorder, such as hepatocellular carcinoma or hepatoblastoma.

Another aspect of the invention features a method of modulating expression, e.g., downregulating or silencing, a target gene, by providing an iRNA agent that has a first sequence and a second sequence sufficiently complementary to each other to hybridize. In addition, the first sequence is complementary to a first target RNA region and the second sequence is complementary to a second target RNA region.

In one embodiment, the iRNA agent is administered to a subject, e.g., a human.

In another embodiment, the first and second sequences are between 15 and 30 nucleotides in length.

In one embodiment, the method of modulating expression of the target gene further includes providing a second iRNA agent that has a third sequence complementary to a third target RNA region. The third sequence can be sufficiently complementary to the first or second sequence to be capable of hybridizing to either the first or second sequence.

Another aspect of the invention features a method of modulating expression, e.g., downregulating or silencing, a plurality of target RNAs, each of the plurality of target RNAs corresponding to a different target gene. The method includes providing an iRNA agent selected by identifying a first region in a first target RNA of the plurality and a second region in a second target RNA of the plurality, where the first and second regions are sufficiently complementary to each other to be capable of hybridizing.

In another aspect of the invention, an iRNA agent molecule includes a first sequence complementary to a first variant RNA target region and a second sequence complementary to a second variant RNA target region, and the first and second variant RNA target regions correspond to first and second variants or mutants of a target gene. In certain embodiments, the target gene is an apoB-100, beta catenin, or glucose-6 phosphatase gene.

In one embodiment, the target gene is a viral gene (e.g., an HCV gene), tumor suppressor or oncogene.

In another embodiment, the first and second variant target RNA regions include allelic variants of the target gene.

In another embodiment, the first and second variant RNA target regions comprise mutations (e.g., point mutations) or polymorphisms of the target gene.

In one embodiment, the first and second variant RNA target regions correspond to hot-spots for genetic variation.

Another aspect of the invention features a plurality (e.g., a panel or bank) of iRNA agents. Each of the iRNA agents of the plurality includes a first sequence complementary to a first variant target RNA region and a second sequence complementary to a second variant target RNA region, where the first and second variant target RNA regions correspond to first and second variants of a target gene. In certain embodiments, the variants are allelic variants of the target gene.

Another aspect of the invention provides a method of identifying an iRNA agent for treating a subject. The method includes providing or obtaining information, e.g., a genotype, about a target gene, providing or obtaining information about a plurality (e.g., panel or bank) of iRNA agents, comparing the information about the target gene to information about the plurality of iRNA agents, and selecting one or more of the plurality of iRNA agents for treating the subject. Each of the plurality of iRNA agents includes a first sequence complementary to a first variant target RNA region and a second sequence complementary to a second variant target RNA region, and the first and second variant target RNA regions correspond to first and second variants of the target gene. The target gene can be an endogenous gene of the subject or a viral gene. The information about the plurality of iRNA agents can be the sequence of the first or second sequence of one or more of the plurality.

In certain embodiments, at least one of the selected iRNA agents includes a sequence capable of hybridizing to an RNA region corresponding to the target gene, and at least one of the selected iRNA agents comprises a sequence capable of hybridizing to an RNA region corresponding to a variant or mutant of the target gene.

In one aspect, the invention relates to compositions and methods for silencing genes expressed in the liver, e.g., to treat disorders of or related to the liver. An iRNA agent composition of the invention can be one which has been modified to alter distribution in favor of the liver.

In another aspect, the invention relates to iRNA agents that can target more than one RNA region, and methods of using and making the iRNA agents. In one embodiment, the RNA is from a gene that is active in the liver, e.g., apoB-100, glucose-6-phosphatase, beta-catenin, or Hepatitis C virus (HCV).

In another aspect, an iRNA agent includes a first and second sequence that are sufficiently complementary to each other to hybridize. The first sequence can be complementary to a first target RNA region and the second sequence can be complementary to a second target RNA region. For example, the first sequence can be complementary to a first target apoB-100 RNA region and the second sequence can be complementary to a second target apoB-100 RNA region.

In one embodiment, the first target RNA region is encoded by a first gene, e.g., a gene expressed in the liver, and the second target RNA region is encoded by a second gene, e.g., a second gene expressed in the liver. In another embodiment, the first and second target RNA regions are different regions of an RNA from a single gene, e.g., a single gene that is at least expressed in the liver. In another embodiment, the first and second sequences differ by at least one and no more than six nucleotides.

In another embodiment, sequence analysis, e.g., to identify common mutants in the target gene, is used to identify a region of the target gene that has high variability or mutational frequency. For example, sequence analysis can be used to identify regions of aopB-100 or beta catenin that have high variability or mutational frequency. In another embodiment, the region of the target gene having high variability or mutational frequency is identified by obtaining or providing genotype information about the target gene from a population. In particular, the genotype information can be from a population suffering from a liver disorder, such as hepatocellular carcinoma or hepatoblastoma.

In another aspect, the invention features a method for reducing apoB-100 levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets apoB-100. The iRNA agent can be one described here, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the apoB-100 gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Tables 5 and 6. In a preferred embodiment it targets both sequences of a palindromic pair provided in Tables 5 and 6. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Tables 5 and 6.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Tables 5 and 6. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Tables 5 and 6 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Tables 5 and 6 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

The iRNA agent that targets apoB-100 can be administered in an amount sufficient to reduce expression of apoB-100 mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of apoB-100 protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%). Preferably, the iRNA agent does not reduce expression of apoB-48 mRNA or protein. This can be effected, e.g., by selection of an iRNA agent which specifically targets the nucleotides subject to RNA editing in the apoB-100 transcript.

The iRNA agent that targets apoB-100 can be administered to a subject, wherein the subject is suffering from a disorder characterized by elevated or otherwise unwanted expression of apoB-100, elevated or otherwise unwanted levels of cholesterol, and/or disregulation of lipid metabolism. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder. These disorders include HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), acquired hyperlipidemia; hypercholestorolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD) coronary heart disease (CHD) atherosclerosis. In one embodiment, the iRNA that targets apoB-100 is administered to a subject suffering from statin-resistant hypercholesterolemia.

The apoB-100 iRNA agent can be administered in an amount sufficient to reduce levels of serum LDL-C and/or HDL-C and/or total cholesterol in a subject. For example, the iRNA is administered in an amount sufficient to decrease total cholesterol by at least 0.5%, 1%, 2.5%, 5%, 10% in the subject. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce the risk of myocardial infarction the subject.

In a preferred embodiment the iRNA agent is administered repeatedly. Administration of an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending on such factors as the severity of a patient's symptoms. For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.

In one embodiment, the iRNA agent can be targeted to the liver, and apoB expression level are decreased in the liver following administration of the apoB iRNA agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to a lipophilic moiety, e.g., a lipid, cholesterol, oleyl, retinyl, or cholesteryl residue (see Table 1). Other lipophilic moieties that can be associated, e.g., conjugated with the iRNA agent include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine. In one embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a low-density lipoprotein (LDL), e.g., a lactosylated LDL. In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a polymeric carrier complex with sugar residues.

In another embodiment, the iRNA agent can be targeted to the liver by associating, e.g., conjugating, the iRNA agent to a liposome complexed with sugar residues. A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver (see Table 1). In a preferred embodiment, the targeting moiety includes more than one galactose moiety, preferably two or three. Preferably, the targeting moiety includes 3 galactose moieties, e.g., spaced about 15 angstroms from each other. The targeting moiety can be lactose. A lactose is a glucose coupled to a galactose. Preferably, the targeting moiety includes three lactoses.

The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose, or mannose-6-phosphate targeting moiety can be used for macrophage targeting. The targeting agent can be linked directly, e.g., covalently or non covalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.

It is particularly preferred to use an iRNA conjugated to a lipophilic molecule to conjugate to an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.

In one embodiment, the iRNA agent has been modified, or is associated with a delivery agent, e.g., a delivery agent described herein, e.g., a liposome, which has been modified to alter distribution in favor of the liver. In one embodiment, the modification mediates association with a serum albumin (SA), e.g., a human serum albumin (HSA), or a fragment thereof.

The iRNA agent, particularly an iRNA agent that targets apoB, beta-catenin or glucose-6-phosphatase RNA, can be targeted to the liver, for example by associating, e.g., conjugating the iRNA agent to an SA molecule, e.g., an HSA molecule, or a fragment thereof. In one embodiment, the iRNA agent or composition thereof has an affinity for an SA, e.g., HSA, which is sufficiently high such that its levels in the liver are at least 10, 20, 30, 50, or 100% greater in the presence of SA, e.g., HSA, or is such that addition of exogenous SA will increase delivery to the liver. These criteria can be measured, e.g., by testing distribution in a mouse in the presence or absence of exogenous mouse or human SA.

The SA, e.g., HSA, targeting agent can be linked directly, e.g., covalently or non-covalently, to the iRNA agent, or to another delivery or formulation modality, e.g., a liposome. E.g., the iRNA agents with or without a targeting moiety can be incorporated into a delivery modality, e.g., a liposome, with or without a targeting moiety.

It is particularly preferred to use an iRNA conjugated to an SA, e.g., an HSA, molecule wherein the iRNA agent is an apoB, beta-catenin or glucose-6-phosphatase iRNA targeting agent.

In another aspect, the invention features, a method for reducing glucose-6-phosphatase levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent which targets glucose-6-phosphatase. The iRNA agent can be a dsRNA that has a sequence that is substantially identical to a sequence of the glucose-6-phosphatase gene.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 7. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 7. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 7.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 7. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 7 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 7 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

Table 7 refers to sequences from human glucose-6-phosphatase. Table 8 refers to sequences from rat glucose-6-phosphatase. The sequences from table 8 can be used, e.g., in experiments with rats or cultured rat cells.

In a preferred embodiment iRNA agent can have any architecture, e.g., architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurring ribonucleotides can be used in the selected iRNA agent.

The iRNA that targets glucose-6-phosphatase can be administered in an amount sufficient to reduce expression of glucose-6-phosphatase mRNA.

The iRNA that targets glucose-6-phosphatase can be administered to a subject to inhibit hepatic glucose production, for the treatment of glucose-metabolism-related disorders, such as diabetes, e.g., type-2-diabetes mellitus. The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder.

In other embodiments, iRNA agents having sequence similarity to the following genes can also be used to inhibit hepatic glucose production. These other genes include “forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphate locator; glucokinase inhibitory regulatory protein; and phosphoenolpyruvate carboxykinase (PEPCK).

In one embodiment, the iRNA agent can be targeted to the liver, and RNA expression levels of the targeted genes are decreased in the liver following administration of the iRNA agent.

The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of a target gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length

In another aspect, the invention features a method for reducing beta-catenin levels in a subject, e.g., a mammal, such as a human. The method includes administering to a subject an iRNA agent that targets beta-catenin. The iRNA agent can be one described herein, and can be a dsRNA that has a sequence that is substantially identical to a sequence of the beta-catenin gene. The iRNA can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 9. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 9. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 9.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 9. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 9. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 9.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 9. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 9as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 9 but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

The iRNA agent that targets beta-catenin can be administered in an amount sufficient to reduce expression of beta-catenin mRNA. In one embodiment, the iRNA agent is administered in an amount sufficient to reduce expression of beta-catenin protein (e.g., by at least 2%, 4%, 6%, 10%, 15%, 20%).

The iRNA agent that targets beta-catenin can be administered to a subject, wherein the subject is suffering from a disorder characterized by unwanted cellular proliferation in the liver or of liver tissue, e.g., metastatic tissue originating from the liver. Examples include , a benign or malignant disorder, e.g., a cancer, e.g., a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma.

The iRNA agent can be administered to an individual at risk for the disorder to delay onset of the disorder or a symptom of the disorder

In a preferred embodiment the iRNA agent is administered repeatedly. Administration of an iRNA agent can be carried out over a range of time periods. It can be administered daily, once every few days, weekly, or monthly. The timing of administration can vary from patient to patient, depending on such factors as the severity of a patient's symptoms. For example, an effective dose of an iRNA agent can be administered to a patient once a month for an indefinite period of time, or until the patient no longer requires therapy. In addition, sustained release compositions containing an iRNA agent can be used to maintain a relatively constant dosage in the patient's blood.

In one embodiment, the iRNA agent can be targeted to the liver, and beta-catenin expression level are decreased in the liver following administration of the beta-catenin iRNA agent. For example, the iRNA agent can be complexed with a moiety that targets the liver, e.g., an antibody or ligand that binds a receptor on the liver.

In another aspect, the invention provides methods to treat liver disorders, e.g., disorders characterized by unwanted cell proliferation, hematological disorders, disorders characterized by inflammation disorders, and metabolic or viral diseases or disorders of the liver. A proliferation disorder of the liver can be, for example, a benign or malignant disorder, e.g., a cancer, e.g, a hepatocellular carcinoma (HCC), hepatic metastasis, or hepatoblastoma. A hepatic hematology or inflammation disorder can be a disorder involving clotting factors, a complement-mediated inflammation or a fibrosis, for example. Metabolic diseases of the liver can include dyslipidemias, and irregularities in glucose regulation. Viral diseases of the liver can include hepatitis C or hepatitis B. In one embodiment, a liver disorder is treated by administering one or more iRNA agents that have a sequence that is substantially identical to a sequence in a gene involved in the liver disorder.

In one embodiment an iRNA agent to treat a liver disorder has a sequence which is substantially identical to a sequence of the beta-catenin or c-jun gene. In another embodiment, such as for the treatment of hepatitis C or hepatitis B, the iRNA agent can have a sequence that is substantially identical to a sequence of a gene of the hepatitis C virus or the hepatitis B virus, respectively. For example, the iRNA agent can target the 5′ core region of HCV. This region lies just downstream of the ribosomal toe-print straddling the initiator methionine. Alternatively, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

In a preferred embodiment, the subject is treated with an iRNA agent which targets one of the sequences listed in Table 10. In a preferred embodiment it targets both sequences of a palindromic pair provided in Table 10. The most preferred targets are listed in descending order of preferrability, in other words, the more preferred targets are listed earlier in Table 10.

In a preferred embodiment the iRNA agent will include regions, or strands, which are complementary to a pair in Table 10. In a preferred embodiment the iRNA agent will include regions complementary to the palindromic pairs of Table 10 as a duplex region.

In a preferred embodiment the duplex region of the iRNA agent will target a sequence listed in Table 10, but will not be perfectly complementary with the target sequence, e.g., it will not be complementary at at least 1 base pair. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, in total, or per strand, which do not hybridize with the target sequence

In a preferred embodiment the iRNA agent includes overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

In another aspect, an iRNA agent can be administered to modulate blood clotting, e.g., to reduce the tendency to form a blood clot. In a preferred embodiment the iRNA agent targets Factor V expression, preferably in the liver. One or more iRNA agents can be used to target a wild type allele, a mutant allele, e.g., the Leiden Factor V allele, or both. Such administration can be used to treat or prevent venous thrombosis, e.g., deep vein thrombosis or pulmonary embolism, or another disorder caused by elevated or otherwise unwanted expression of Factor V, in, e.g., the liver. In one embodiment the iRNA agent can treat a subject, e.g., a human who has Factor V Leiden or other genetic trait associated with an unwanted tendency to form blood clots.

In a preferred embodiment administration of an iRNA agent which targets Factor V is with the administration of a second treatment, e.g, a treatment which reduces the tendency of the blood to clot, e.g., the administration of heparin or of a low molecular weight heparin.

In one embodiment, the iRNA agent that targets Factor V can be used as a prophylaxis in patients, e.g., patients with Factor V Leiden, who are placed at risk for a thrombosis, e.g., those about to undergo surgery, in particular those about to undergo high-risk surgical procedures known to be associated with formation of venous thrombosis, those about to undergo a prolonged period of relative inactivity, e.g., on a motor vehicle, train or airplane flight, e.g., a flight or other trip lasting more than three or five hours. Such a treatment can be an adjunct to the therapeutic use of low molecular weight (LMW) heparin prophylaxis.

In another embodiment, the iRNA agent that targets Factor V can be administered to patients with Factor V Leiden to treat deep vein thrombosis (DVT) or pulmonary embolism (PE). Such a treatment can be an adjunct to (or can replace) therapeutic uses of heparin or coumadin. The treatment can be administered by inhalation or generally by pulmonary routes.

In a preferred embodiment, an iRNA agent administered to treat a liver disorder is targeted to the liver. For example, the iRNA agent can be complexed with a targeting moiety, e.g., an antibody or ligand that recognizes a liver-specific receptor.

The invention also includes preparations, including substantially pure or pharmaceutically acceptable preparations of iRNA agents which silence any of the genes discussed herein and in particular for any of apoB-100, glucose-6-phosphatase, beta-catenin, factor V, or any of the HVC genes discussed herein.

The methods and compositions of the invention, e.g., the methods and compositions to treat diseases and disorders of the liver described herein, can be used with any of the iRNA agents described. In addition, the methods and compositions of the invention can be used for the treatment of any disease or disorder described herein, and for the treatment of any subject, e.g., any animal, any mammal, such as any human.

In another aspect, the invention features, a method of selecting two sequences or strands for use in an iRNA agent. The method includes:

providing a first candidate sequence and a second candidate sequence;

determining the value of a parameter which is a function of the number of palindromic pairs between the first and second sequence, wherein a palindromic pair is a nucleotide on said first sequence which, when the sequences are aligned in anti-parallel orientation, will hybridize with a nucleotide on said second sequence;

comparing the number with a predetermined reference value, and if the number has a predetermined relationship with the reference, e.g., if it is the same or greater, selecting the sequences for use in an iRNA agent. In most cases each of the two sequences will be completely complementary with a target sequence (though as described elsewhere herein that may not always be the case, there may not be perfect complementarity with one or both of the target sequences) and will have sufficient complementarity with each other to form a duplex. The parameter can be derived e.g., by directly determining the number of palindromic pairs, e.g., by inspection or by the use of a computer program which compares or analyses sequence. The parameter can also be determined less directly, and include e.g., calculation of or measurement of the Tm or other value related to the free energy of association or dissociation of a duplex.

In a preferred embodiment the determination can be performed on a target sequence, e.g., a genomic sequence. In such embodiments the selected sequence is converted to its complement in the iRNA agent.

In a preferred embodiment the first and second sequences are selected from the sequence of a single target gene. In other embodiments the first sequence is selected from the sequence of a first target gene and the second sequence is selected from the target of a second target gene.

In a preferred embodiment the method includes comparing blocks of sequence, e.g., blocks which are between 15 and 25 nucleotides in length, and preferably 19, 20, or 21, and most preferably 19 nucleotides in length, to determine if they are suitable for use, e.g., if they possess sufficient palindromic pairs.

In a preferred embodiment the first and second sequences are divided into a plurality of regions, e.g., terminal regions and a middle region disposed between the terminal regions and where in the reference value, or the predetermined relationship to the reference value, is different for at least two regions. E.g., the first and second sequences, when aligned in anti-parallel orientation, are divided into terminal regions each of a selected number of base pairs, e.g., 2, 3, 4, 5, or 6, and a middle region, and the reference value for the terminal regions is higher than for the middle regions. In other words, a higher number or proportion of palindromic pairs is required in the terminal regions.

In a preferred embodiment the first and second sequences are gene sequences thus the complements of the sequences will be used in a iRNA agent. In a preferred embodiment hybridize means a classical Watson-Crick pairing. In other embodiments hybridize can include non-Watson-Crick paring, e.g., parings seen in micro RNA precursors.

In a preferred embodiment the method includes the addition of nucleotides to form overhangs, e.g., 3′ or 5′ overhangs, preferably one or more 3′ overhangs. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined , e.g., by additional bases to form a hairpin, or by other non-base linkers.

In a preferred embodiment the method is used to select all or part of a iRNA agent. The selected sequences can be incorporated into an iRNA agent having any architecture, e.g., an architecture described herein. E.g., it can be incorporated into an iRNA agent having an overhang structure, overall length, hairpin vs. two-strand structure, as described herein. In addition, monomers other than naturally occurring ribonucleotides can be used in the selected iRNA agent.

Preferred iRNA agents of this method will target genes expressed in the liver, e.g., one of the genes disclosed herein, e.g., apo B, Beta catenin, an HVC gene, or glucose 6 phosphatase.

In another aspect, the invention features, an iRNA agent, determined, made, or selected by a method described herein.

The methods and compositions of the invention, e.g., the methods and iRNA compositions to treat liver-based diseases described herein, can be used with any dosage and/or formulation described herein, as well as with any route of administration described herein.

The invention also provides for the use of an iRNA agent which includes monomers which can form other than a canonical Watson-Crick pairing with another monomer, e.g., a monomer on another strand.

The use of “other than canonical Watson-Crick pairing” between monomers of a duplex can be used to control, often to promote, melting of all or part of a duplex. The iRNA agent can include a monomer at a selected or constrained position that results in a first level of stability in the iRNA agent duplex (e.g., between the two separate molecules of a double stranded iRNA agent) and a second level of stability in a duplex between a sequence of an iRNA agent and another sequence molecule, e.g., a target or off-target sequence in a subject. In some cases the second duplex has a relatively greater level of stability, e.g., in a duplex between an anti-sense sequence of an iRNA agent and a target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and the target sequence (sometimes referred to herein as the selection or constraint parameters), are selected such that the iRNA agent duplex is has a comparatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting disassociation of the duplex iRNA agent in the context of the RISC) while the duplex formed between an anti-sense targeting sequence and its target sequence, has a relatively higher free energy of association (which while not wishing to be bound by mechanism or theory, is believed to contribute to efficacy by promoting association of the anti-sense sequence and the target RNA).

In other cases the second duplex has a relatively lower level of stability, e.g., in a duplex between a sense sequence of an iRNA agent and an off-target mRNA. In this case one or more of the monomers, the position of the monomers in the iRNA agent, and an off-target sequence, are selected such that the iRNA agent duplex is has a comparatively higher free energy of association while the duplex formed between a sense targeting sequence and its off-target sequence, has a relatively lower free energy of association (which while not wishing to be bound by mechanism or theory, is believed to reduce the level of off-target silencing by contribute to efficacy by promoting disassociation of the duplex formed by the sense strand and the off-target sequence).

Thus, inherent in the structure of the iRNA agent is the property of having a first stability for the intra-iRNA agent duplex and a second stability for a duplex formed between a sequence from the iRNA agent and another RNA, e.g., a target mRNA. As discussed above, this can be accomplished by judicious selection of one or more of the monomers at a selected or constrained position, the selection of the position in the duplex to place the selected or constrained position, and selection of the sequence of a target sequence (e.g., the particular region of a target gene which is to be targeted). The iRNA agent sequences which satisfy these requirements are sometimes referred herein as constrained sequences. Exercise of the constraint or selection parameters can be, e.g., by inspection, or by computer assisted methods. Exercise of the parameters can result in selection of a target sequence and of particular monomers to give a desired result in terms of the stability, or relative stability, of a duplex.

Thus, in one aspect, the invention features, an iRNA agent which includes: a first sequence which targets a first target region and a second sequence which targets a second target region. The first and second sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the first target region has a first monomer, and the second target region has a second monomer. The first and second monomers occupy complementary or corresponding positions. One, and preferably both monomers are selected such that the stability of the pairing of the monomers contribute to a duplex between the first and second sequence will differ form the stability of the pairing between the first or second sequence with a target sequence.

Usually, the monomers will be selected (selection of the target sequence may be required as well) such that they form a pairing in the iRNA agent duplex which has a lower free energy of dissociation, and a lower Tm, than will be possessed by the paring of the monomer with its complementary monomer in a duplex between the iRNA agent sequence and a target RNA duplex.

The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.

The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.

E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

The monomers can be selected such that: first and second monomers are naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites either do not pair and have no substantial level of H-bonding, or form a non canonical Watson-Crick pairing and form a non-canonical pattern of H bonding, which usually have a lower free energy of dissociation than seen in a canonical Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one (or both) of the iRNA agent sequences duplexes with a target, the first (or second) monomer forms a canonical Watson-Crick pairing with the base in the complementary position on the target, or forms a non canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. The classical Watson-Crick parings are as follows: A-T, G-C, and A-U. Non-canonical Watson-Crick pairings are known in the art and can include, U-U, G-G, G-Atrans, G-Acis, and GU.

The monomer in one or both of the sequences is selected such that, it does not pair, or forms a pair with its corresponding monomer in the other sequence which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the one sequence and its monomer at the corresponding site in the other sequence are less stable than the H bonds formed by the monomer one (or both) of the sequences with the respective target sequence. The monomer in one or both strands is also chosen to promote stability in one or both of the duplexes made by a strand and its target sequence. E.g., one or more of the monomers and the target sequences are selected such that at the selected or constrained position, there is are no H bonds formed, or a non canonical pairing is formed in the iRNA agent duplex, or otherwise they otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing, but when one (or both) sequences form a duplex with the respective target, the pairing at the selected or constrained site is a canonical Watson-Crick pairing.

The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the a sequence and the intended target will not be destabilized.

By way of example:

the monomer at the selected site in the first sequence includes an A (or a modified base which pairs with T), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

the monomer at the selected site in the first sequence includes U (or a modified base which pairs with A), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

The monomer at the selected site in the first sequence includes a G (or a modified base which pairs with C), and the monomer in at the selected position in the second sequence is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., G, Acis, Atrans, or U. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

The monomer at the selected site in the first sequence includes a C (or a modified base which pairs with G), and the monomer in at the selected position in the second sequence is chosen a monomer which will not pair or which will form a non-canonical pairing. These will be useful in applications wherein the target sequence for the first sequence has a T at the selected position. In embodiments where both target duplexes are stabilized it is useful wherein the target sequence for the second strand has a monomer which will form a canonical Watson-Crick pairing with the monomer selected for the selected position in the second strand.

In another embodiment a non-naturally occurring or modified monomer or monomers are chosen such that when a non-naturally occurring or modified monomer occupies a positions at the selected or constrained position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T.

When placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable. This is shown in the FIG. 1.

The pair shown in FIG. 1 (the 2-amino A and the 2-s U and T) is exemplary. In another embodiment, the monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An example of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. (Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. Examples can also be found in the section on Universal Bases below.) In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes: a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a target gene in a subject. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In a duplex region of the iRNA agent, at a selected or constrained position, the monomers are selected such that:

the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability (e.g., the H bonding formed between the monomer at the selected site in the sense strand and its monomer at the corresponding site in the anti-sense strand are less stable than the H bonds formed by the monomer of the anti-sense sequence and its canonical Watson-Crick partner or, if the monomer in the anti-sense strand includes a modified base, the natural analog of the modified base and its canonical Watson-Crick partner);

the monomer is in the corresponding position in the anti-sense strand is selected such that it maximizes the stability of a duplex it forms with the target sequence, e.g., it forms a canonical Watson-Crick paring with the monomer in the corresponding position on the target stand;

optionally, the monomer in the sense sequence is selected such that, it does not pair, or forms a pair with its corresponding monomer in the anti-sense strand which minimizes stability with an off-target sequence.

The inclusion of such a monomers will have one or more of the following effects: it will destabilize the iRNA agent duplex, it will destabilize interactions between the sense sequence and unintended target sequences, sometimes referred to as off-target sequences, and duplex interactions between the anti-sense strand and the intended target will not be destabilized.

The constraint placed upon the monomers can be applied at a selected site or at more than one selected site. By way of example, the constraint can be applied at more than 1, but less than 3, 4, 5, 6, or 7 sites in an iRNA agent duplex.

A constrained or selected site can be present at a number of positions in the iRNA agent duplex. E.g., a constrained or selected site can be present within 3, 4, 5, or 6 positions from either end, 3′ or 5′ of a duplexed sequence. A constrained or selected site can be present in the middle of the duplex region, e.g., it can be more than 3, 4, 5, or 6, positions from the end of a duplexed region.

The iRNA agent can be selected to target a broad spectrum of genes, including any of the genes described herein.

In a preferred embodiment the iRNA agent has an architecture (architecture refers to one or more of overall length, length of a duplex region, the presence, number, location, or length of overhangs, sing strand versus double strand form) described herein.

E.g., the iRNA agent can be less than 30 nucleotides in length, e.g., 21-23 nucleotides. Preferably, the iRNA is 21 nucleotides in length and there is a duplex region of about 19 pairs. In one embodiment, the iRNA is 21 nucleotides in length, and the duplex region of the iRNA is 19 nucleotides. In another embodiment, the iRNA is greater than 30 nucleotides in length.

In some embodiment the duplex region of the iRNA agent will have, mismatches, in addition to the selected or constrained site or sites. Preferably it will have no more than 1, 2, 3, 4, or 5 bases, which do not form canonical Watson-Crick pairs or which do not hybridize. Overhangs are discussed in detail elsewhere herein but are preferably about 2 nucleotides in length. The overhangs can be complementary to the gene sequences being targeted or can be other sequence. TT is a preferred overhang sequence. The first and second iRNA agent sequences can also be joined, e.g., by additional bases to form a hairpin, or by other non-base linkers.

One or more selection or constraint parameters can be exercised such that: monomers at the selected site in the sense and anti-sense sequences are both naturally occurring ribonucleotides, or modified ribonucleotides having naturally occurring bases, and when occupying complementary sites in the iRNA agent duplex either do not pair and have no substantial level of H-bonding, or form a non-canonical Watson-Crick pairing and thus form a non-canonical pattern of H bonding, which generally have a lower free energy of dissociation than seen in a Watson-Crick pairing, or otherwise pair to give a free energy of association which is less than that of a preselected value or is less, e.g., than that of a canonical pairing. When one, usually the anti-sense sequence of the iRNA agent sequences forms a duplex with another sequence, generally a sequence in the subject, and generally a target sequence, the monomer forms a classic Watson-Crick pairing with the base in the complementary position on the target, or forms a non-canonical Watson-Crick pairing having a higher free energy of dissociation and a higher Tm than seen in the paring in the iRNA agent. Optionally, when the other sequence of the iRNA agent, usually the sense sequences forms a duplex with another sequence, generally a sequence in the subject, and generally an off-target sequence, the monomer fails to forms a canonical Watson-Crick pairing with the base in the complementary position on the off target sequence, e.g., it forms or forms a non-canonical Watson-Crick pairing having a lower free energy of dissociation and a lower Tm.

By way of example:

the monomer at the selected site in the anti-sense stand includes an A (or a modified base which pairs with T), the corresponding monomer in the target is a T, and the sense strand is chosen from a base which will not pair or which will form a noncanonical pair, e.g., G;

the monomer at the selected site in the anti-sense stand includes a U (or a modified base which pairs with A), the corresponding monomer in the target is an A, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing, e.g., U or G;

the monomer at the selected site in the anti-sense stand includes a C (or a modified base which pairs with G), the corresponding monomer in the target is a G, and the sense strand is chosen a monomer which will not pair or which will form a non-canonical pairing, e.g., G, A_(cis), A_(trans), or U; or

the monomer at the selected site in the anti-sense stand includes a G (or a modified base which pairs with C), the corresponding monomer in the target is a C, and the sense strand is chosen from a monomer which will not pair or which will form a non-canonical pairing.

In another embodiment a non-naturally occurring or modified monomer or monomers is chosen such that when it occupies complementary a position in an iRNA agent they exhibit a first free energy of dissociation and when one (or both) of them pairs with a naturally occurring monomer, the pair exhibits a second free energy of dissociation, which is usually higher than that of the pairing of the first and second monomers. E.g., when the first and second monomers occupy complementary positions they either do not pair and have no substantial level of H-bonding, or form a weaker bond than one of them would form with a naturally occurring monomer, and reduce the stability of that duplex, but when the duplex dissociates at least one of the strands will form a duplex with a target in which the selected monomer will promote stability, e.g., the monomer will form a more stable pair with a naturally occurring monomer in the target sequence than the pairing it formed in the iRNA agent.

An example of such a pairing is 2-amino A and either of a 2-thio pyrimidine analog of U or T. As is discussed above, when placed in complementary positions of the iRNA agent these monomers will pair very poorly and will minimize stability. However, a duplex is formed between 2 amino A and the U of a naturally occurring target, or a duplex is formed between 2-thio U and the A of a naturally occurring target or 2-thio T and the A of a naturally occurring target will have a relatively higher free energy of dissociation and be more stable.

The monomer at the selected position in the sense strand can be a universal pairing moiety. A universal pairing agent will form some level of H bonding with more than one and preferably all other naturally occurring monomers. An examples of a universal pairing moiety is a monomer which includes 3-nitro pyrrole. Examples of other candidate universal base analogs can be found in the art, e.g., in Loakes, 2001, NAR 29: 2437-2447, hereby incorporated by reference. In these cases the monomer at the corresponding position of the anti-sense strand can be chosen for its ability to form a duplex with the target and can include, e.g., A, U, G, or C.

In another aspect, the invention features, an iRNA agent which includes: a sense sequence, which preferably does not target a sequence in a subject, and an anti-sense sequence, which targets a plurality of target sequences in a subject, wherein the targets differ in sequence at only 1 or a small number, e.g., no more than 5, 4, 3 or 2 positions. The sense and anti-sense sequences have sufficient complementarity to each other to hybridize, e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme. In the sequence of the anti-sense strand of the iRNA agent is selected such that at one, some, or all of the positions which correspond to positions that differ in sequence between the target sequences, the anti-sense strand will include a monomer which will form H-bonds with at least two different target sequences. In a preferred example the anti-sense sequence will include a universal or promiscuous monomer, e.g., a monomer which includes 5-nitro pyrrole, 2-amino A, 2-thio U or 2-thio T, or other universal base referred to herein.

In a preferred embodiment the iRNA agent targets repeated sequences (which differ at only one or a small number of positions from each other) in a single gene, a plurality of genes, or a viral genome, e.g., the HCV genome.

An embodiment is illustrated in the FIGS. 2 and 3.

In another aspect, the invention features, determining, e.g., by measurement or calculation, the stability of a pairing between monomers at a selected or constrained position in the iRNA agent duplex, and preferably determining the stability for the corresponding pairing in a duplex between a sequence form the iRNA agent and another RNA, e.g., a target sequence. The determinations can be compared. An iRNA agent thus analyzed can be used in the development of a further modified iRNA agent or can be administered to a subject. This analysis can be performed successively to refine or design optimized iRNA agents.

In another aspect, the invention features, a kit which includes one or more of the following an iRNA described herein, a sterile container in which the iRNA agent is disclosed, and instructions for use.

In another aspect, the invention features, an iRNA agent containing a constrained sequence made by a method described herein. The iRNA agent can target one or more of the genes referred to herein.

iRNA agents having constrained or selected sites, e.g., as described herein, can be used in any way described herein. Accordingly, they iRNA agents having constrained or selected sites, e.g., as described herein, can be used to silence a target, e.g., in any of the methods described herein and to target any of the genes described herein or to treat any of the disorders described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be incorporated into any of the formulations or preparations, e.g., pharmaceutical or sterile preparations described herein. iRNA agents having constrained or selected sites, e.g., as described herein, can be administered by any of the routes of administration described herein.

The term “other than canonical Watson-Crick pairing” as used herein, refers to a pairing between a first monomer in a first sequence and a second monomer at the corresponding position in a second sequence of a duplex in which one or more of the following is true: (1) there is essentially no pairing between the two, e.g., there is no significant level of H bonding between the monomers or binding between the monomers does not contribute in any significant way to the stability of the duplex; (2) the monomers are a non-canonical paring of monomers having a naturally occurring bases, i.e., they are other than A-T, A-U, or G-C, and they form monomer-monomer H bonds, although generally the H bonding pattern formed is less strong than the bonds formed by a canonical pairing; or (3) at least one of the monomers includes a non-naturally occurring bases and the H bonds formed between the monomers is, preferably formed is less strong than the bonds formed by a canonical pairing, namely one or more of A-T, A-U, G-C.

The term “off-target” as used herein, refers to a sequence other than the sequence to be silenced.

Universal Bases: “wild-cards”; shape-based complementarity

Bi-stranded, multisite replication of a base pair between difluorotoluene and adenine: confirmation by ‘inverse’ sequencing. Liu, D.; Moran, S.; Kool, E. T. Chem. Biol., 1997, 4, 919-926)

(Importance of terminal base pair hydrogen-bonding in 3′-end proofreading by the Klenow fragment of DNA polymerase I. Morales, J. C.; Kool, E. T. Biochemistry, 2000, 39, 2626-2632)

(Selective and stable DNA base pairing without hydrogen bonds. Matray, T, J.; Kool, E. T. J. Am. Chem. Soc., 1998, 120, 6191-6192)

(Difluorotoluene, a nonpolar isostere for thymine, codes specifically and efficiently for adenine in DNA replication. Moran, S. Ren, R. X.-F.; Rumney IV, S.; Kool, E. T. J. Am. Chem. Soc., 1997, 119, 2056-2057)

(Structure and base pairing properties of a replicable nonpolar isostere for deoxyadenosine. Guckian, K. M.; Morales, J. C.; Kool, E. T. J. Org. Chem., 1998, 63, 9652-9656)

(Universal bases for hybridization, replication and chain termination. Berger, M.; Wu. Y.; Ogawa, A. K.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. Nucleic Acids Res., 2000, 28, 2911-2914)

(1. Efforts toward the expansion of the genetic alphabet: Information storage and replication with unnatural hydrophobic base pairs. Ogawa, A. K.; Wu, Y.; McMinn, D. L.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 3274-3287. 2. Rational design of an unnatural base pair with increased kinetic selectivity. Ogawa, A. K.; Wu. Y.; Berger, M.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 8803-8804)

(Efforts toward expansion of the genetic alphabet: replication of DNA with three base pairs. Tae, E. L.; Wu, Y.; Xia, G.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2001, 123, 7439-7440)

(1. Efforts toward expansion of the genetic alphabet: Optimization of interbase hydrophobic interactions. Wu, Y.; Ogawa, A. K.; Berger, M.; McMinn, D. L.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 2000, 122, 7621-7632. 2. Efforts toward expansion of genetic alphabet: DNA polymerase recognition of a highly stable, self-pairing hydrophobic base. McMinn, D. L.; Ogawa. A. K.; Wu, Y.; Liu, J.; Schultz, P. G.; Romesberg, F. E. J. Am. Chem. Soc., 1999, 121, 11585-11586)

(A stable DNA duplex containing a non-hydrogen-bonding and non-shape complementary base couple: Interstrand stacking as the stability determining factor. Brotschi, C.; Haberli, A.; Leumann, C, J. Angew. Chem. Int. Ed., 2001, 40, 3012-3014)

(2,2′-Bipyridine Ligandoside: A novel building block for modifying DNA with intra-duplex metal

complexes. Weizman, H ; Tor, Y. J. Am. Chem. Soc., 2001, 123, 3375-3376)

(Minor groove hydration is critical to the stability of DNA duplexes. Lan, T.; McLaughlin, L. W. J. Am. Chem. Soc., 2000, 122, 6512-13)

(Effect of the Universal base 3-nitropyrrole on the selectivity of neighboring natural bases. Oliver, J. S.; Parker, K. A.; Suggs, J. W. Organic Lett., 2001, 3, 1977-1980. 2. Effect of the 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrol residue on the stability of DNA duplexes and triplexes. Amosova, O.; George J.; Fresco, J. R. Nucleic Acids Res., 1997, 25, 1930-1934. 3. Synthesis, structure and deoxyribonucleic acid sequencing with a universal nucleosides: 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole. Bergstrom, D. E.; Zhang, P.; Toma, P. H.; Andrews, P. C.; Nichols, R. J. Am. Chem. Soc., 1995, 117, 1201-1209)

(Model studies directed toward a general triplex DNA recognition scheme: a novel DNA base that binds a CG base-pair in an organic solvent. Zimmerman, S. C.; Schmitt, P. J. Am. Chem. Soc., 1995, 117, 10769-10770)

(A universal, photocleavable DNA base: nitropiperonyl 2′-deoxyriboside. J. Org. Chem., 2001, 66, 2067-2071)

(Recognition of a single guanine bulge by 2-acylamino-1,8-naphthyridine. Nakatani, K ; Sando, S.; Saito, I. J. Am. Chem. Soc., 2000, 122, 2172-2177. b. Specific binding of 2-amino-1,8-naphthyridine into single guanine bulge as evidenced by photooxidation of GC doublet, Nakatani, K ; Sando, S.; Yoshida, K.; Saito, I. Bioorg. Med. Chem. Lett., 2001, 11, 335-337)

Other universal bases can have the following formulas:

wherein:

Q is N or CR⁴⁴;

Q′ is N or CR⁴⁵;

Q″ is N or CR⁴⁷;

Q′″ is N or CR⁴⁹;

Q^(iv) is N or CR⁵⁰;

R⁴⁴ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c), C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁵ forms —OCH₂O—;

R⁴⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c) , C₁-C₆ alkyl, C₆-C₁₀aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁴ or R⁴⁶ forms —OCH₂O—;

R⁴⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c) , C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁵ or R⁴⁷ forms —OCH₂O—;

R⁴⁷ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c) , C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁶ or R⁴⁸ forms —OCH2O—;

R⁴⁸ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c) , C₁-C₆ alkyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, or when taken together with R⁴⁷ forms —OCH2O—;

R⁴⁹, R⁵⁰, R⁵¹, R⁵², R⁵³, R⁵⁴, R⁵⁷, R⁵⁸, R⁵⁹, R⁶⁰, R⁶¹, R⁶², R⁶³, R⁶⁴, R⁶⁵, R⁶⁶, R⁶⁷, R⁶⁸, R⁶⁹, R⁷⁰, R⁷¹, and R⁷² are each independently selected from hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c) , C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o);

R⁵⁵ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c) , C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o), or when taken together with R⁵⁶ forms a fused aromatic ring which may be optionally substituted;

R⁵⁶ is hydrogen, halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c) , C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o) , or when taken together with R⁵⁵ forms a fused aromatic ring which may be optionally substituted;

R¹⁷ is halo, NH₂, NHR^(b), or NR^(b)R^(c) ;

R^(b) is C₁-C₆ alkyl or a nitrogen protecting group;

R^(c) is C₁-C₆ alkyl; and

R^(o) is alkyl optionally substituted with halo, hydroxy, nitro, protected hydroxy, NH₂, NHR^(b), or NR^(b)R^(c) , C₁-C₆ alkyl, C₂-C₆ alkynyl, C₆-C₁₀ aryl, C₆-C₁₀ heteroaryl, C₃-C₈ heterocyclyl, NC(O)R¹⁷, or NC(O)R^(o) .

Examples of universal bases include:

In one aspect, the invention features methods of producing iRNA agents, e.g., sRNA agents, e.g. an sRNA agent described herein, having the ability to mediate RNAi. These iRNA agents can be formulated for administration to a subject.

In another aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.

In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days.

In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.

The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser that delivers a metered dose. Selected modes of delivery are discussed in more detail below.

The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.

In some embodiments the medication is delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.

In one embodiment, the rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution.

In another embodiment, the rectal administration is by means of a suppository. The suppository can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.

The invention also provides methods, compositions, and kits for oral delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA described herein, e.g., a iRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered orally.

Oral administration can be in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. In a preferred embodiment the composition is applied topically to a surface of the oral cavity.

The invention also provides methods, compositions, and kits for buccal delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to the buccal cavity. The medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

The invention also provides methods, compositions, and kits for ocular delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to ocular tissue.

The medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. It can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser that delivers a metered dose.

The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure.

Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

The invention also provides methods, compositions, and kits for delivery of iRNA agents described herein to or through the skin.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a

DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and one or two 1-3 nucleotide single strand 3′ overhangs can be administered directly to the skin.

The medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue.

In one embodiment, the administration of the iRNA agent composition is topical. In another embodiment, topical administration delivers the composition to the dermis or epidermis of a subject. In other embodiments the topical administration is in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly.

In another embodiment, the transdermal administration is applied with at least one penetration enhancer. In other embodiments, the penetration can be enhanced with iontophoresis, phonophoresis, and sonophoresis. In another aspect, the invention provides methods, compositions, devices, and kits for pulmonary delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a

DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent, e.g., a sRNA agent having a double stranded region of less than 40, preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication.

The preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.

In another aspect, the invention provides methods, compositions, devices, and kits for nasal delivery of iRNA agents described herein. Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of iRNA agent, e.g., a sRNA agent having a double stranded region of less than 40 and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication.

The preferred method of nasal delivery is by spray, aerosol, liquid, e.g., by drops, of by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser which delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.

In another embodiment, the iRNA agent is packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

In one aspect, of the invention, the dosage of a pharmaceutical composition including a iRNA agent is administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease.

In another aspect, gene expression in a subject is modulated by administering a pharmaceutical composition including a iRNA agent. In other embodiments, a subject is treated with the pharmaceutical composition by any of the methods mentioned above. In another embodiment, the subject has cancer.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition can be administered as a liposome. For example, the composition can be prepared by a method that includes: (1) contacting a iRNA agent with an amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex. In one embodiment, the detergent is cholate, deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS (3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane), novel-4-D-glucopyranoside, lauryl dimethylamine oxide, or octylglucoside. The iRNA agent can be an sRNA agent. The method can include preparing a composition that includes a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In another aspect, a subject is treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form. In one embodiment, the powder is a collection of microparticles. In one embodiment, the powder is a collection of crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In one aspect, a subject is treated by administering a defined amount of a iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In one aspect, the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), is provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. In one embodiment, this includes providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.

In another aspect, a subject is treated for a condition treatable by inhalation. In one embodiment, this method includes aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

In another aspect, the invention features a method of treating a subject that includes: administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques

In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load,)

In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.

In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes.

In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof

In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.

In one aspect, the invention features a method for making a pharmaceutical composition, the method including: (1) contacting an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) with a amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex.

In another aspect, the invention features a pharmaceutical composition produced by a method including: (1) contacting an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) with a amphipathic cationic lipid conjugate in the presence of a detergent; and (2) removing the detergent to form a iRNA agent and cationic lipid complex. In one embodiment, the detergent is cholate, deoxycholate, lauryl sarcosine, octanoyl sucrose, CHAPS (3-[(3-cholamidopropyl)-di-methylamine]-2-hydroxyl-1-propane), novel-∂-D-glucopyranoside, lauryl dimethylamine oxide, or octylglucoside. In another embodiment, the amphipathic cationic lipid conjugate is biodegradable. In yet another embodiment the pharmaceutical composition includes a targeting ligand.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form. In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof. The microsphere can be formulated as a suspension, a powder, or an implantable solid.

In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, a-lactalbumin, trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns.

In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having the structure or characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.

As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The materials, methods, and examples are illustrative only and not intended to be limiting. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, useful methods and materials are described below. Other features and advantages of the invention will be apparent from the accompanying drawings and description, and from the claims. The contents of all references, pending patent applications and published patents, cited throughout this application are hereby expressly incorporated by reference. In case of conflict, the present specification, including definitions, will control.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a structural representation of base pairing in psuedocomplementary siRNA².

FIG. 2 is a schematic representation of dual targeting siRNAs (SEQ ID NOs 3952, 3953, 3954, and 3955 respectively) designed to target the HCV genome.

FIG. 3 is a schematic representation of pseudocomplementary, bifunctional siRNAs (SEQ ID NOs 3956, 3957, 3958, and 4087 respectively) designed to target the HCV genome.

FIG. 4 is a general synthetic scheme for incorporation of RRMS monomers into an oligonucleotide.

FIG. 5 is a table of representative RRMS carriers. Panel 1 shows pyrroline-based RRMSs; panel 2 shows 3-hydroxyproline-based RRMSs; panel 3 shows piperidine-based

RRMSs; panel 4 shows morpholine and piperazine-based RRMSs; and panel 5 shows decalin-based RRMSs. R¹ is succinate or phosphoramidate and R² is H or a conjugate ligand.

FIG. 6A is a graph depicting levels of luciferase mRNA in livers of CMV-Luc mice (Xanogen) following intervenous injection (iv) of buffer or siRNA into the tail vein. Each bar represents data from one mouse. RNA levels were quantified by QuantiGene Assay (Genospectra, Inc.; Fremont, Calif.)). The Y axis represents chemiluminescence values in counts per second (CPS).

FIG. 6B is a graph depicting levels of luciferase mRNA in livers of CMV-Luc mice (Xanogen). The values are averaged from the data depicted in FIG. 6A.

FIG. 7 is a graph depicting the pharmacokinetics of cholesterol-conjugated and unconjugated siRNA. The diamonds represent the amount of unconjugated ³³P-labeled siRNA (ALN-3000) in mouse plasma over time; the squares represent the amount of cholesterol-conjugated ³³P-labeled siRNA (ALN-3001) in mouse plasma over time. “L1163” is equivalent to ALN3000; “L1163Chol” is equivalent to ALN-3001.

DETAILED DESCRIPTION

Double-stranded (dsRNA) directs the sequence-specific silencing of mRNA through a process known as RNA interference (RNAi). The process occurs in a wide variety of organisms, including mammals and other vertebrates.

It has been demonstrated that 21-23 nt fragments of dsRNA are sequence-specific mediators of RNA silencing, e.g., by causing RNA degradation. While not wishing to be bound by theory, it may be that a molecular signal, which may be merely the specific length of the fragments, present in these 21-23 nt fragments recruits cellular factors that mediate RNAi. Described herein are methods for preparing and administering these 21-23 nt fragments, and other iRNAs agents, and their use for specifically inactivating gene function. The use of iRNAs agents (or recombinantly produced or chemically synthesized oligonucleotides of the same or similar nature) enables the targeting of specific mRNAs for silencing in mammalian cells. In addition, longer dsRNA agent fragments can also be used, e.g., as described below.

Although, in mammalian cells, long dsRNAs can induce the interferon response which is frequently deleterious, sRNAs do not trigger the interferon response, at least not to an extent that is deleterious to the cell and host. In particular, the length of the iRNA agent strands in an sRNA agent can be less than 31, 30, 28, 25, or 23 nt, e.g., sufficiently short to avoid inducing a deleterious interferon response. Thus, the administration of a composition of sRNA agent (e.g., formulated as described herein) to a mammalian cell can be used to silence expression of a target gene while circumventing the interferon response. Further, use of a discrete species of iRNA agent can be used to selectively target one allele of a target gene, e.g., in a subject heterozygous for the allele.

Moreover, in one embodiment, a mammalian cell is treated with an iRNA agent that disrupts a component of the interferon response, e.g., double stranded RNA (dsRNA)-activated protein kinase PKR. Such a cell can be treated with a second iRNA agent that includes a sequence complementary to a target RNA and that has a length that might otherwise trigger the interferon response.

In a typical embodiment, the subject is a mammal such as a cow, horse, mouse, rat, dog, pig, goat, or a primate. The subject can be a dairy mammal (e.g., a cow, or goat) or other farmed animal (e.g., a chicken, turkey, sheep, pig, fish, shrimp). In a much preferred embodiment, the subject is a human, e.g., a normal individual or an individual that has, is diagnosed with, or is predicted to have a disease or disorder.

Further, because iRNA agent mediated silencing persists for several days after administering the iRNA agent composition, in many instances, it is possible to administer the composition with a frequency of less than once per day, or, for some instances, only once for the entire therapeutic regimen. For example, treatment of some cancer cells may be mediated by a single bolus administration, whereas a chronic viral infection may require regular administration, e.g., once per week or once per month.

A number of exemplary routes of delivery are described that can be used to administer an iRNA agent to a subject. In addition, the iRNA agent can be formulated according to an exemplary method described herein.

iRNA Agent Structure

Described herein are isolated iRNA agents, e.g., RNA molecules, (double-stranded; single-stranded) that mediate RNAi. The iRNA agents preferably mediate RNAi with respect to an endogenous gene of a subject or to a gene of a pathogen.

An “RNA agent” as used herein, is an unmodified RNA, modified RNA, or nucleoside surrogate, all of which are defined herein (see, e.g., the section below entitled RNA Agents). While numerous modified RNAs and nucleoside surrogates are described, preferred examples include those which have greater resistance to nuclease degradation than do unmodified RNAs. Preferred examples include those which have a 2′ sugar modification, a modification in a single strand overhang, preferably a 3′ single strand overhang, or, particularly if single stranded, a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

An “iRNA agent” as used herein, is an RNA agent which can, or which can be cleaved into an RNA agent which can, down regulate the expression of a target gene, preferably an endogenous or pathogen target RNA. While not wishing to be bound by theory, an iRNA agent may act by one or more of a number of mechanisms, including post-transcriptional cleavage of a target mRNA sometimes referred to in the art as RNAi, or pre-transcriptional or pre-translational mechanisms. An iRNA agent can include a single strand or can include more than one strands, e.g., it can be a double stranded iRNA agent. If the iRNA agent is a single strand it is particularly preferred that it include a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group.

The iRNA agent should include a region of sufficient homology to the target gene, and be of sufficient length in terms of nucleotides, such that the iRNA agent, or a fragment thereof, can mediate down regulation of the target gene. (For ease of exposition the term nucleotide or ribonucleotide is sometimes used herein in reference to one or more monomeric subunits of an RNA agent. It will be understood herein that the usage of the term “ribonucleotide” or “nucleotide”, herein can, in the case of a modified RNA or nucleotide surrogate, also refer to a modified nucleotide, or surrogate replacement moiety at one or more positions.) Thus, the iRNA agent is or includes a region which is at least partially, and in some embodiments fully, complementary to the target RNA. It is not necessary that there be perfect complementarity between the iRNA agent and the target, but the correspondence must be sufficient to enable the iRNA agent, or a cleavage product thereof, to direct sequence specific silencing, e.g., by RNAi cleavage of the target RNA, e.g., mRNA.

Complementarity, or degree of homology with the target strand, is most critical in the antisense strand. While perfect complementarity, particularly in the antisense strand, is often desired some embodiments can include, particularly in the antisense strand, one or more but preferably 6, 5, 4, 3, 2, or fewer mismatches (with respect to the target RNA). The mismatches, particularly in the antisense strand, are most tolerated in the terminal regions and if present are preferably in a terminal region or regions, e.g., within 6, 5, 4, or 3 nucleotides of the 5′ and/or 3′ terminus. The sense strand need only be sufficiently complementary with the antisense strand to maintain the over all double strand character of the molecule.

As discussed elsewhere herein, an iRNA agent will often be modified or include nucleoside surrogates in addition to the RRMS. Single stranded regions of an iRNA agent will often be modified or include nucleoside surrogates, e.g., the unpaired region or regions of a hairpin structure, e.g., a region which links two complementary regions, can have modifications or nucleoside surrogates. Modification to stabilize one or more 3′- or 5′-terminus of an iRNA agent, e.g., against exonucleases, or to favor the antisense sRNA agent to enter into RISC are also favored. Modifications can include C3 (or C6, C7, C12) amino linkers, thiol linkers, carboxyl linkers, non-nucleotidic spacers (C3, C6, C9, C12, abasic, triethylene glycol, hexaethylene glycol), special biotin or fluorescein reagents that come as phosphoramidites and that have another DMT-protected hydroxyl group, allowing multiple couplings during RNA synthesis.

iRNA agents include: molecules that are long enough to trigger the interferon response (which can be cleaved by Dicer (Bernstein et al. 2001. Nature, 409:363-366) and enter a RISC (RNAi-induced silencing complex)); and, molecules which are sufficiently short that they do not trigger the interferon response (which molecules can also be cleaved by Dicer and/or enter a RISC), e.g., molecules which are of a size which allows entry into a RISC, e.g., molecules which resemble Dicer-cleavage products. Molecules that are short enough that they do not trigger an interferon response are termed sRNA agents or shorter iRNA agents herein. “sRNA agent or shorter iRNA agent” as used herein, refers to an iRNA agent, e.g., a double stranded RNA agent or single strand agent, that is sufficiently short that it does not induce a deleterious interferon response in a human cell, e.g., it has a duplexed region of less than 60 but preferably less than 50, 40, or 30 nucleotide pairs. The sRNA agent, or a cleavage product thereof, can down regulate a target gene, e.g., by inducing RNAi with respect to a target RNA, preferably an endogenous or pathogen target RNA.

Each strand of an sRNA agent can be equal to or less than 30, 25, 24, 23, 22, 21, or 20 nucleotides in length. The strand is preferably at least 19 nucleotides in length. For example, each strand can be between 21 and 25 nucleotides in length. Preferred sRNA agents have a duplex region of 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, and one or more overhangs, preferably one or two 3′ overhangs, of 2- 3 nucleotides.

In addition to homology to target RNA and the ability to down regulate a target gene, an iRNA agent will preferably have one or more of the following properties:

(1) it will be of the Formula 1, 2, 3, or 4 set out in the RNA Agent section below;

(2) if single stranded it will have a 5′ modification which includes one or more phosphate groups or one or more analogs of a phosphate group;

(3) it will, despite modifications, even to a very large number, or all of the nucleosides, have an anti sense strand that can present bases (or modified bases) in the proper three dimensional framework so as to be able to form correct base pairing and form a duplex structure with a homologous target RNA which is sufficient to allow down regulation of the target, e.g., by cleavage of the target RNA;

(4) it will, despite modifications, even to a very large number, or all of the nucleosides, still have “RNA-like” properties, i.e., it will possess the overall structural, chemical and physical properties of an RNA molecule, even though not exclusively, or even partly, of ribonucleotide-based content. For example, an iRNA agent can contain, e.g., a sense and/or an antisense strand in which all of the nucleotide sugars contain e.g., 2′ fluoro in place of 2′ hydroxyl. This deoxyribonucleotide-containing agent can still be expected to exhibit RNA-like properties. While not wishing to be bound by theory, the electronegative fluorine prefers an axial orientation when attached to the C2′ position of ribose. This spatial preference of fluorine can, in turn, force the sugars to adopt a C₃′-endo pucker. This is the same puckering mode as observed in RNA molecules and gives rise to the RNA-characteristic A-family-type helix. Further, since fluorine is a good hydrogen bond acceptor, it can participate in the same hydrogen bonding interactions with water molecules that are known to stabilize RNA structures. (Generally, it is preferred that a modified moiety at the 2′ sugar position will be able to enter into H-bonding which is more characteristic of the OH moiety of a ribonucleotide than the H moiety of a deoxyribonucleotide. A preferred iRNA agent will: exhibit a C_(3′)-endo pucker in all, or at least 50, 75,80, 85, 90, or 95% of its sugars; exhibit a C_(3′)-endo pucker in a sufficient amount of its sugars that it can give rise to a the RNA-characteristic A-family-type helix; will have no more than 20, 10, 5, 4, 3, 2, orl sugar which is not a C_(3′)-endo pucker structure. These limitations are particularly preferably in the antisense strand;

(5) regardless of the nature of the modification, and even though the RNA agent can contain deoxynucleotides or modified deoxynucleotides, particularly in overhang or other single strand regions, it is preferred that DNA molecules, or any molecule in which more than 50, 60, or 70% of the nucleotides in the molecule, or more than 50, 60, or 70% of the nucleotides in a duplexed region are deoxyribonucleotides, or modified deoxyribonucleotides which are deoxy at the 2′ position, are excluded from the definition of RNA agent.

A “single strand iRNA agent” as used herein, is an iRNA agent which is made up of a single molecule. It may include a duplexed region, formed by intra-strand pairing, e.g., it may be, or include, a hairpin or pan-handle structure. Single strand iRNA agents are preferably antisense with regard to the target molecule. In preferred embodiments single strand iRNA agents are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxygen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-). (These modifications can also be used with the antisense strand of a double stranded iRNA.)

A single strand iRNA agent should be sufficiently long that it can enter the RISC and participate in RISC mediated cleavage of a target mRNA. A single strand iRNA agent is at least 14, and more preferably at least 15, 20, 25, 29, 35, 40, or 50nucleotides in length. It is preferably less than 200, 100, or 60 nucleotides in length.

Hairpin iRNA agents will have a duplex region equal to or at least 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs. The duplex region will preferably be equal to or less than 200, 100, or 50, in length. Preferred ranges for the duplex region are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. The hairpin will preferably have a single strand overhang or terminal unpaired region, preferably the 3′, and preferably of the antisense side of the hairpin. Preferred overhangs are 2-3 nucleotides in length.

A “double stranded (ds) iRNA agent” as used herein, is an iRNA agent which includes more than one, and preferably two, strands in which interchain hybridization can form a region of duplex structure.

The antisense strand of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length.

The sense strand of a double stranded iRNA agent should be equal to or at least 14, 15, 16 17, 18, 19, 25, 29, 40, or 60 nucleotides in length. It should be equal to or less than 200, 100, or 50, nucleotides in length. Preferred ranges are 17 to 25, 19 to 23, and 19 to21 nucleotides in length.

The double strand portion of a double stranded iRNA agent should be equal to or at least, 14, 15, 16 17, 18, 19, 20, 21, 22, 23, 24, 25, 29, 40, or 60 nucleotide pairs in length. It should be equal to or less than 200, 100, or 50, nucleotides pairs in length. Preferred ranges are 15-30, 17 to 23, 19 to 23, and 19 to 21 nucleotides pairs in length. 2653

In many embodiments, the ds iRNA agent is sufficiently large that it can be cleaved by an endogenous molecule, e.g., by Dicer, to produce smaller ds iRNA agents, e.g., sRNAs agents

It may be desirable to modify one or both of the antisense and sense strands of a double strand iRNA agent. In some cases they will have the same modification or the same class of modification but in other cases the sense and antisense strand will have different modifications, e.g., in some cases it is desirable to modify only the sense strand. It may be desirable to modify only the sense strand, e.g., to inactivate it, e.g., the sense strand can be modified in order to inactivate the sense strand and prevent formation of an active sRNA/protein or RISC. This can be accomplished by a modification which prevents 5′-phosphorylation of the sense strand, e.g., by modification with a 5′-O-methyl ribonucleotide (see Nykanen et al., (2001) ATP requirements and small interfering RNA structure in the RNA interference pathway. Cell 107, 309-321.) Other modifications which prevent phosphorylation can also be used, e.g., simply substituting the 5′-OH by H rather than O-Me. Alternatively, a large bulky group may be added to the 5′-phosphate turning it into a phosphodiester linkage, though this may be less desirable as phosphodiesterases can cleave such a linkage and release a functional sRNA 5′-end. Antisense strand modifications include 5′ phosphorylation as well as any of the other 5′ modifications discussed herein, particularly the 5′ modifications discussed above in the section on single stranded iRNA molecules. It is preferred that the sense and antisense strands be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains sense and antisense strands, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.

Preferred lengths for the duplexed region is between 15 and 30, most preferably 18, 19, 20, 21, 22, and 23 nucleotides in length, e.g., in the sRNA agent range discussed above. sRNA agents can resemble in length and structure the natural Dicer processed products from long dsRNAs. Embodiments in which the two strands of the sRNA agent are linked, e.g., covalently linked are also included. Hairpin, or other single strand structures which provide the required double stranded region, and preferably a 3′ overhang are also within the invention.

The isolated iRNA agents described herein, including ds iRNA agents and sRNA agents can mediate silencing of a target RNA, e.g., mRNA, e.g., a transcript of a gene that encodes a protein. For convenience, such mRNA is also referred to herein as mRNA to be silenced. Such a gene is also referred to as a target gene. In general, the RNA to be silenced is an endogenous gene or a pathogen gene. In addition, RNAs other than mRNA, e.g., tRNAs, and viral RNAs, can also be targeted.

As used herein, the phrase “mediates RNAi” refers to the ability to silence, in a sequence specific manner, a target RNA. While not wishing to be bound by theory, it is believed that silencing uses the RNAi machinery or process and a guide RNA, e.g., an sRNA agent of 21 to 23 nucleotides.

As used herein, “specifically hybridizable” and “complementary” are terms which are used to indicate a sufficient degree of complementarity such that stable and specific binding occurs between a compound of the invention and a target RNA molecule. Specific binding requires a sufficient degree of complementarity to avoid non-specific binding of the oligomeric compound to non-target sequences under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays or therapeutic treatment, or in the case of in vitro assays, under conditions in which the assays are performed. The non-target sequences typically differ by at least 5 nucleotides.

In one embodiment, an iRNA agent is “sufficiently complementary” to a target RNA, e.g., a target mRNA, such that the iRNA agent silences production of protein encoded by the target mRNA. In another embodiment, the iRNA agent is “exactly complementary” (excluding the RRMS containing subunit(s))to a target RNA, e.g., the target RNA and the iRNA agent anneal, preferably to form a hybrid made exclusively of Watson-Crick basepairs in the region of exact complementarity. A “sufficiently complementary” target RNA can include an internal region (e.g., of at least 10 nucleotides) that is exactly complementary to a target RNA. Moreover, in some embodiments, the iRNA agent specifically discriminates a single-nucleotide difference. In this case, the iRNA agent only mediates RNAi if exact complementary is found in the region (e.g., within 7 nucleotides of) the single-nucleotide difference.

As used herein, the term “oligonucleotide” refers to a nucleic acid molecule (RNA or DNA) preferably of length less than 100, 200, 300, or 400 nucleotides.

RNA agents discussed herein include otherwise unmodified RNA as well as RNA which have been modified, e.g., to improve efficacy, and polymers of nucleoside surrogates. Unmodified RNA refers to a molecule in which the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are the same or essentially the same as that which occur in nature, preferably as occur naturally in the human body. The art has referred to rare or unusual, but naturally occurring, RNAs as modified RNAs, see, e.g., Limbach et al., (1994) Summary: the modified nucleosides of RNA, Nucleic Acids Res. 22: 2183-2196. Such rare or unusual RNAs, often termed modified RNAs (apparently because the are typically the result of a post transcriptionally modification) are within the term unmodified RNA, as used herein. Modified RNA as used herein refers to a molecule in which one or more of the components of the nucleic acid, namely sugars, bases, and phosphate moieties, are different from that which occur in nature, preferably different from that which occurs in the human body. While they are referred to as modified “RNAs,” they will of course, because of the modification, include molecules which are not RNAs. Nucleoside surrogates are molecules in which the ribophosphate backbone is replaced with a non-ribophosphate construct that allows the bases to the presented in the correct spatial relationship such that hybridization is substantially similar to what is seen with a ribophosphate backbone, e.g., non-charged mimics of the ribophosphate backbone. Examples of all of the above are discussed herein.

Much of the discussion below refers to single strand molecules. In many embodiments of the invention a double stranded iRNA agent, e.g., a partially double stranded iRNA agent, is required or preferred. Thus, it is understood that that double stranded structures (e.g. where two separate molecules are contacted to form the double stranded region or where the double stranded region is formed by intramolecular pairing (e.g., a hairpin structure)) made of the single stranded structures described below are within the invention. Preferred lengths are described elsewhere herein.

As nucleic acids are polymers of subunits or monomers, many of the modifications described below occur at a position which is repeated within a nucleic acid, e.g., a modification of a base, or a phosphate moiety, or the a non-linking O of a phosphate moiety. In some cases the modification will occur at all of the subject positions in the nucleic acid but in many, and infact in most cases it will not. By way of example, a modification may only occur at a 3′ or 5′ terminal position, may only occur in a terminal regions, e.g. at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand. A modification may occur in a double strand region, a single strand region, or in both. A modification may occur only in the double strand region of an RNA or may only occur in a single strand region of an RNA. E.g., a phosphorothioate modification at a non-linking O position may only occur at one or both termini, may only occur in a terminal regions, e.g., at a position on a terminal nucleotide or in the last 2, 3, 4, 5, or 10 nucleotides of a strand, or may occur in double strand and single strand regions, particularly at termini. The 5′ end or ends can be phosphorylated.

In some embodiments it is particularly preferred, e.g., to enhance stability, to include particular bases in overhangs, or to include modified nucleotides or nucleotide surrogates, in single strand overhangs, e.g., in a 5′ or 3′ overhang, or in both. E.g., it can be desirable to include purine nucleotides in overhangs. In some embodiments all or some of the bases in a 3′ or 5′ overhang will be modified, e.g., with a modification described herein. Modifications can include, e.g., the use of modifications at the 2′ OH group of the ribose sugar, e.g., the use of deoxyribonucleotides, e.g., deoxythymidine, instead of ribonucleotides, and modifications in the phosphate group, e.g., phosphothioate modifications. Overhangs need not be homologous with the target sequence.

Modifications and nucleotide surrogates are discussed below.

The scaffold presented above in Formula 1 represents a portion of a ribonucleic acid. The basic components are the ribose sugar, the base, the terminal phosphates, and phosphate internucleotide linkers. Where the bases are naturally occurring bases, e.g., adenine, uracil, guanine or cytosine, the sugars are the unmodified 2′ hydroxyl ribose sugar (as depicted) and W, X, Y, and Z are all O, Formula 1 represents a naturally occurring unmodified oligoribonucleotide.

Unmodified oligoribonucleotides may be less than optimal in some applications, e.g., unmodified oligoribonucleotides can be prone to degradation by e.g., cellular nucleases. Nucleases can hydrolyze nucleic acid phosphodiester bonds. However, chemical modifications to one or more of the above RNA components can confer improved properties, and, e.g., can render oligoribonucleotides more stable to nucleases. Umodified oligoribonucleotides may also be less than optimal in terms of offering tethering points for attaching ligands or other moieties to an iRNA agent.

Modified nucleic acids and nucleotide surrogates can include one or more of:

(i) alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens (When the phosphate is in the terminal position, one of the positions W or Z will not link the phosphate to an additional element in a naturally occurring ribonucleic acid. However, for simplicity of terminology, except where otherwise noted, the W position at the 5′ end of a nucleic acid and the terminal Z position at the 3′ end of a nucleic acid, are within the term “linking phosphate oxygens” as used herein.);

(ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2′ hydroxyl on the ribose sugar, or wholesale replacement of the ribose sugar with a structure other than ribose, e.g., as described herein;

(iii) wholesale replacement of the phosphate moiety (bracket I) with “dephospho” linkers;

(iv) modification or replacement of a naturally occurring base;

(v) replacement or modification of the ribose-phosphate backbone (bracket II);

(vi) modification of the 3′ end or 5′ end of the RNA, e.g., removal, modification or replacement of a terminal phosphate group or conjugation of a moiety, e.g. a fluorescently labeled moiety, to either the 3′ or 5′ end of RNA.

The terms replacement, modification, alteration, and the like, as used in this context, do not imply any process limitation, e.g., modification does not mean that one must start with a reference or naturally occurring ribonucleic acid and modify it to produce a modified ribonucleic acid bur rather modified simply indicates a difference from a naturally occurring molecule.

It is understood that the actual electronic structure of some chemical entities cannot be adequately represented by only one canonical form (i.e. Lewis structure). While not wishing to be bound by theory, the actual structure can instead be some hybrid or weighted average of two or more canonical forms, known collectively as resonance forms or structures. Resonance structures are not discrete chemical entities and exist only on paper. They differ from one another only in the placement or “localization” of the bonding and nonbonding electrons for a particular chemical entity. It can be possible for one resonance structure to contribute to a greater extent to the hybrid than the others. Thus, the written and graphical descriptions of the embodiments of the present invention are made in terms of what the art recognizes as the predominant resonance form for a particular species. For example, any phosphoroamidate (replacement of a nonlinking oxygen with nitrogen) would be represented by X═O and Y═N in the above figure.

Specific modifications are discussed in more detail below.

The Phosphate Group

The phosphate group is a negatively charged species. The charge is distributed equally over the two non-linking oxygen atoms (i.e., X and Y in Formula 1 above). However, the phosphate group can be modified by replacing one of the oxygens with a different substituent. One result of this modification to RNA phosphate backbones can be increased resistance of the oligoribonucleotide to nucleolytic breakdown. Thus while not wishing to be bound by theory, it can be desirable in some embodiments to introduce alterations which result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

Examples of modified phosphate groups include phosphorothioate, phosphoroselenates, borano phosphates, borano phosphate esters, hydrogen phosphonates, phosphoroamidates, alkyl or aryl phosphonates and phosphotriesters. Phosphorodithioates have both non-linking oxygens replaced by sulfur. Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of oligoribonucleotides diastereomers. Diastereomer formation can result in a preparation in which the individual diastereomers exhibit varying resistance to nucleases. Further, the hybridization affinity of RNA containing chiral phosphate groups can be lower relative to the corresponding unmodified RNA species. Thus, while not wishing to be bound by theory, modifications to both X and Y which eliminate the chiral center, e.g. phosphorodithioate formation, may be desirable in that they cannot produce diastereomer mixtures. Thus, X can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Thus Y can be any one of S, Se, B, C, H, N, or OR (R is alkyl or aryl). Replacement of X and/or Y with sulfur is preferred.

The phosphate linker can also be modified by replacement of a linking oxygen (i.e., W or Z in Formula 1) with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at a terminal oxygen (position W (3′) or position Z (5′). Replacement of W with carbon or Z with nitrogen is preferred.

Candidate agents can be evaluated for suitability as described below.

The Sugar Group

A modified RNA can include modification of all or some of the sugar groups of the ribonucleic acid. E.g., the 2′ hydroxyl group (OH) can be modified or replaced with a number of different “oxy” or “deoxy” substituents. While not being bound by theory, enhanced stability is expected since the hydroxyl can no longer be deprotonated to form a 2′ alkoxide ion. The 2′ alkoxide can catalyze degradation by intramolecular nucleophilic attack on the linker phosphorus atom. Again, while not wishing to be bound by theory, it can be desirable to some embodiments to introduce alterations in which alkoxide formation at the 2′ position is not possible.

Examples of “oxy”-2′ hydroxyl group modifications include alkoxy or aryloxy (OR, e.g., R═H, alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar); polyethyleneglycols (PEG), O(CH₂CH₂O)_(n)CH₂CH₂OR; “locked” nucleic acids (LNA) in which the 2′ hydroxyl is connected, e.g., by a methylene bridge, to the 4′ carbon of the same ribose sugar; O-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino) and aminoalkoxy, O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino). It is noteworthy that oligonucleotides containing only the methoxyethyl group (MOE), (OCH₂CH₂OCH₃, a PEG derivative), exhibit nuclease stabilities comparable to those modified with the robust phosphorothioate modification.

“Deoxy” modifications include hydrogen (i.e. deoxyribose sugars, which are of particular relevance to the overhang portions of partially ds RNA); halo (e.g., fluoro); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); NH(CH₂CH₂NH)—CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino), —NHC(O)R (R=alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino functionality. Preferred substitutents are 2′-methoxyethyl, 2′-OCH3,2′-O-allyl, 2′-C- allyl, and 2′-fluoro.

The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified RNA can include nucleotides containing e.g., arabinose, as the sugar.

Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. These abasic sugars can also be further contain modifications at one or more of the constituent sugar atoms.

To maximize nuclease resistance, the 2′ modifications can be used in combination with one or more phosphate linker modifications (e.g., phosphorothioate). The so-called “chimeric” oligonucleotides are those that contain two or more different modifications.

The modificaton can also entail the wholesale replacement of a ribose structure with another entity at one or more sites in the iRNA agent. These modifications are described in section entitled Ribose Replacements for RRMSs.

Candidate modifications can be evaluated as described below.

Replacement of the Phosphate Group

The phosphate group can be replaced by non-phosphorus containing connectors (cf. Bracket I in Formula 1 above). While not wishing to be bound by theory, it is believed that since the charged phosphodiester group is the reaction center in nucleolytic degradation, its replacement with neutral structural mimics should impart enhanced nuclease stability. Again, while not wishing to be bound by theory, it can be desirable, in some embodiment, to introduce alterations in which the charged phosphate group is replaced by a neutral moiety.

Examples of moieties which can replace the phosphate group include siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methyl enedimethylhydrazo and methyleneoxymethylimino. Preferred replacements include the methylenecarbonylamino and methylenemethylimino groups.

Candidate modifications can be evaluated as described below.

Replacement of Ribophosphate Backbone

Oligonucleotide- mimicking scaffolds can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates (see Bracket II of Formula 1 above). While not wishing to be bound by theory, it is believed that the absence of a repetitively charged backbone diminishes binding to proteins that recognize polyanions (e.g. nucleases). Again, while not wishing to be bound by theory, it can be desirable in some embodiment, to introduce alterations in which the bases are tethered by a neutral surrogate backbone.

Examples include the mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates. A preferred surrogate is a PNA surrogate.

Candidate modifications can be evaluated as described below.

Terminal Modifications

The 3′ and 5′ ends of an oligonucleotide can be modified. Such modifications can be at the 3′ end, 5′ end or both ends of the molecule. They can include modification or replacement of an entire terminal phosphate or of one or more of the atoms of the phosphate group. E.g., the 3′ and 5′ ends of an oligonucleotide can be conjugated to other functional molecular entities such as labeling moieties, e.g., fluorophores (e.g., pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes) or protecting groups (based e.g., on sulfur, silicon, boron or ester). The functional molecular entities can be attached to the sugar through a phosphate group and/or a spacer. The terminal atom of the spacer can connect to or replace the linking atom of the phosphate group or the C-3′ or C-5′ O, N, S or C group of the sugar. Alternatively, the spacer can connect to or replace the terminal atom of a nucleotide surrogate (e.g., PNAs). These spacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents. When a spacer/phosphate-functional molecular entity-spacer/phosphate array is interposed between two strands of iRNA agents, this array can substitute for a hairpin RNA loop in a hairpin-type RNA agent. The 3′ end can be an —OH group. While not wishing to be bound by theory, it is believed that conjugation of certain moieties can improve transport, hybridization, and specificity properties. Again, while not wishing to be bound by theory, it may be desirable to introduce terminal alterations that improve nuclease resistance. Other examples of terminal modifications include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic carriers (e.g., cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles).

Terminal modifications can be added for a number of reasons, including as discussed elsewhere herein to modulate activity or to modulate resistance to degradation. Terminal modifications useful for modulating activity include modification of the 5′ end with phosphate or phosphate analogs. E.g., in preferred embodiments iRNA agents, especially antisense strands, are 5′ phosphorylated or include a phosphoryl analog at the 5′ prime terminus. 5′-phosphate modifications include those which are compatible with RISC mediated gene silencing. Suitable modifications include: 5′-monophosphate ((HO)2(O)P—O-5′); 5′-diphosphate ((HO)2(O)P—O—P(HO)(O)—O-5′); 5′-triphosphate ((HO)2(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′); 5′-monothiophosphate (phosphorothioate; (HO)2(S)P—O-5′); 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)2(O)P—S-5′); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)2(O)P—NH-5′, (HO)(NH2)(O)P—O-5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)2(O)P-5′-CH2-), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH2-), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-).

Terminal modifications useful for increasing resistance to degradation include

Terminal modifications can also be useful for monitoring distribution, and in such cases the preferred groups to be added include fluorophores, e.g., fluorscein or an Alexa dye, e.g., Alexa 488. Terminal modifications can also be useful for enhancing uptake, useful modifications for this include cholesterol. Terminal modifications can also be useful for cross-linking an RNA agent to another moiety; modifications useful for this include mitomycin C.

Candidate modifications can be evaluated as described below.

The Bases

Adenine, guanine, cytosine and uracil are the most common bases found in RNA. These bases can be modified or replaced to provide RNA's having improved properties. E.g., nuclease resistant oligoribonucleotides can be prepared with these bases or with synthetic and natural nucleobases (e.g., inosine, thymine, xanthine, hypoxanthine, nubularine, isoguanisine, or tubercidine) and any one of the above modifications.

Alternatively, substituted or modified analogs of any of the above bases, e.g., “unusual bases” and “universal bases,” can be employed. Examples include without limitation 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyl adenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases. Further purines and pyrimidines include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in the Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, and those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613.

Generally, base changes are less preferred for promoting stability, but they can be useful for other reasons, e.g., some, e.g., 2,6-diaminopurine and 2 amino purine, are fluorescent. Modified bases can reduce target specificity. This should be taken into consideration in the design of iRNA agents.

Candidate modifications can be evaluated as described below.

Evaluation of Candidate RNA's

One can evaluate a candidate RNA agent, e.g., a modified RNA, for a selected property by exposing the agent or modified molecule and a control molecule to the appropriate conditions and evaluating for the presence of the selected property. For example, resistance to a degradent can be evaluated as follows. A candidate modified RNA (and preferably a control molecule, usually the unmodified form) can be exposed to degradative conditions, e.g., exposed to a milieu, which includes a degradative agent, e.g., a nuclease. E.g., one can use a biological sample, e.g., one that is similar to a milieu, which might be encountered, in therapeutic use, e.g., blood or a cellular fraction, e.g., a cell-free homogenate or disrupted cells. The candidate and control could then be evaluated for resistance to degradation by any of a number of approaches. For example, the candidate and control could be labeled, preferably prior to exposure, with, e.g., a radioactive or enzymatic label, or a fluorescent label, such as Cy3 or Cy5. Control and modified RNA's can be incubated with the degradative agent, and optionally a control, e.g., an inactivated, e.g., heat inactivated, degradative agent. A physical parameter, e.g., size, of the modified and control molecules are then determined. They can be determined by a physical method, e.g., by polyacrylamide gel electrophoresis or a sizing column, to assess whether the molecule has maintained its original length, or assessed functionally. Alternatively, Northern blot analysis can be used to assay the length of an unlabeled modified molecule.

A functional assay can also be used to evaluate the candidate agent. A functional assay can be applied initially or after an earlier non-functional assay, (e.g., assay for resistance to degradation) to determine if the modification alters the ability of the molecule to silence gene expression. For example, a cell, e.g., a mammalian cell, such as a mouse or human cell, can be co-transfected with a plasmid expressing a fluorescent protein, e.g., GFP, and a candidate RNA agent homologous to the transcript encoding the fluorescent protein (see, e.g., WO 00/44914). For example, a modified dsRNA homologous to the GFP mRNA can be assayed for the ability to inhibit GFP expression by monitoring for a decrease in cell fluorescence, as compared to a control cell, in which the transfection did not include the candidate dsRNA, e.g., controls with no agent added and/or controls with a non-modified RNA added. Efficacy of the candidate agent on gene expression can be assessed by comparing cell fluorescence in the presence of the modified and unmodified dsRNA agents.

In an alternative functional assay, a candidate dsRNA agent homologous to an endogenous mouse gene, preferably a maternally expressed gene, such as c-mos, can be injected into an immature mouse oocyte to assess the ability of the agent to inhibit gene expression in vivo (see, e.g., WO 01/36646). A phenotype of the oocyte, e.g., the ability to maintain arrest in metaphase II, can be monitored as an indicator that the agent is inhibiting expression. For example, cleavage of c-mos mRNA by a dsRNA agent would cause the oocyte to exit metaphase arrest and initiate parthenogenetic development (Colledge et al. Nature 370: 65-68, 1994; Hashimoto et al. Nature, 370:68-71, 1994). The effect of the modified agent on target RNA levels can be verified by Northern blot to assay for a decrease in the level of target mRNA, or by Western blot to assay for a decrease in the level of target protein, as compared to a negative control. Controls can include cells in which with no agent is added and/or cells in which a non-modified RNA is added.

References

General References

The oligoribonucleotides and oligoribonucleosides used in accordance with this invention may be with solid phase synthesis, see for example “Oligonucleotide synthesis, a practical approach”, Ed. M. J. Gait, IRL Press, 1984; “Oligonucleotides and Analogues, A Practical Approach”, Ed. F. Eckstein, IRL Press, 1991 (especially Chapter 1, Modern machine-aided methods of oligodeoxyribonucleotide synthesis, Chapter 2, Oligoribonucleotide synthesis, Chapter 3, 2′-O-Methyloligoribonucleotide-s: synthesis and applications, Chapter 4, Phosphorothioate oligonucleotides, Chapter 5, Synthesis of oligonucleotide phosphorodithioates, Chapter 6, Synthesis of oligo-2′-deoxyribonucleoside methylphosphonates, and. Chapter 7, Oligodeoxynucleotides containing modified bases. Other particularly useful synthetic procedures, reagents, blocking groups and reaction conditions are described in Martin, P., Helv. Chim. Acta, 1995, 78, 486-504; Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1992, 48, 2223-2311 and Beaucage, S. L. and Iyer, R. P., Tetrahedron, 1993, 49, 6123-6194, or references referred to therein.

Modification described in WO 00/44895, WO01/75164, or WO02/44321 can be used herein.

The disclosure of all publications, patents, and published patent applications listed herein are hereby incorporated by reference.

Phosphate Group References

The preparation of phosphinate oligoribonucleotides is described in U.S. Pat. No. 5,508,270. The preparation of alkyl phosphonate oligoribonucleotides is described in U.S. Pat. No. 4,469,863. The preparation of phosphoramidite oligoribonucleotides is described in U.S. Pat. No. 5,256,775 or U.S. Pat. No. 5,366,878. The preparation of phosphotriester oligoribonucleotides is described in U.S. Pat. No. 5,023,243. The preparation of borano phosphate oligoribonucleotide is described in U.S. Pat. Nos. 5,130,302 and 5,177,198. The preparation of 3′-Deoxy-3′-amino phosphoramidate oligoribonucleotides is described in U.S. Pat. No. 5,476,925. 3′-Deoxy-3′-methylenephosphonate oligoribonucleotides is described in An, H, et al. J. Org. Chem. 2001, 66, 2789-2801. Preparation of sulfur bridged nucleotides is described in Sproat et al. Nucleosides Nucleotides 1988, 7,651 and Crosstick et al. Tetrahedron Lett. 1989, 30, 4693.

Sugar Group References

Modifications to the 2′ modifications can be found in Verma, S. et al. Annu. Rev. Biochem. 1998, 67, 99-134 and all references therein. Specific modifications to the ribose can be found in the following references: 2′-fluoro (Kawasaki et. al., J. Med. Chem., 1993, 36, 831-841), 2′-MOE (Martin, P. Helv. Chim. Acta 1996, 79, 1930-1938), “LNA” (Wengel, J. Acc. Chem. Res. 1999, 32, 301-310).

Replacement of the Phosphate Group References

Methylenemethylimino linked oligoribonucleosides, also identified herein as MMI linked oligoribonucleosides, methylenedimethylhydrazo linked oligoribonucleosides, also identified herein as MDH linked oligoribonucleosides, and methylenecarbonylamino linked oligonucleosides, also identified herein as amide-3 linked oligoribonucleosides, and methyleneaminocarbonyl linked oligonucleosides, also identified herein as amide-4 linked oligoribonucleosides as well as mixed backbone compounds having, as for instance, alternating MMI and PO or PS linkages can be prepared as is described in U.S. Pat. Nos. 5,378,825, 5,386,023, 5,489,677 and in published PCT applications PCT/US92/04294 and PCT/US92/04305 (published as WO 92/20822 WO and 92/20823, respectively). Formacetal and thioformacetal linked oligoribonucleosides can be prepared as is described in U.S. Pat. Nos. 5,264,562 and 5,264,564. Ethylene oxide linked oligoribonucleosides can be prepared as is described in U.S. Pat. No. 5,223,618. Siloxane replacements are described in Cormier, J. F. et al. Nucleic Acids Res. 1988, 16, 4583. Carbonate replacements are described in Tittensor, J. R. J. Chem. Soc. C 1971, 1933. Carboxymethyl replacements are described in Edge, M. D. et al. J. Chem. Soc. Perkin Trans. 1 1972, 1991. Carbamate replacements are described in Stirchak, E. P. Nucleic Acids Res. 1989, 17, 6129.

Replacement of the Phosphate-Ribose Backbone References

Cyclobutyl sugar surrogate compounds can be prepared as is described in U.S. Pat. No. 5,359,044. Pyrrolidine sugar surrogate can be prepared as is described in U.S. Pat. No. 5,519,134. Morpholino sugar surrogates can be prepared as is described in U.S. Pat. Nos. 5,142,047 and 5,235,033, and other related patent disclosures. Peptide Nucleic Acids (PNAs) are known per se and can be prepared in accordance with any of the various procedures referred to in Peptide Nucleic Acids (PNA): Synthesis, Properties and Potential Applications, Bioorganic & Medicinal Chemistry, 1996, 4, 5-23. They may also be prepared in accordance with U.S. Pat. No. 5,539,083.

Terminal Modification References

Terminal modifications are described in Manoharan, M. et al. Antisense and Nucleic Acid Drug Development 12, 103-128 (2002) and references therein.

Bases References

N-2 substitued purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,459,255. 3-Deaza purine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,457,191. 5,6-Substituted pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,614,617. 5-Propynyl pyrimidine nucleoside amidites can be prepared as is described in U.S. Pat. No. 5,484,908. Additional references can be disclosed in the above section on base modifications.

Preferred iRNA Agents

Preferred RNA agents have the following structure (see Formula 2 below):

Referring to Formula 2 above, R¹, R², and R³ are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5 -uracil (pseudouracil), 4-thiouracil, 5 -halouracil, 5 -(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil, substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methyl cytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyl adenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

R⁴, R⁵, and R⁶ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸; O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR²; N(R⁸)₂; NH(CH₂CH₂NH)_(m)CH₂CH₂NHR⁹; NHC(O)R⁸; cyano; mercapto, SR⁸; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, or ureido; or R⁴, R⁵, or R⁶ together combine with R⁷ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

A¹ is:

; H; OH; OCH₃; W¹; an abasic nucleotide; or absent;

(a preferred A1, especially with regard to anti-sense strands, is chosen from 5′-monophosphate ((HO)₂(O)P—O-5′), 5′-diphosphate ((HO)₂(O)P—O—P(HO)(O)—O-5′), 5′-triphosphate ((HO)₂(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-guanosine cap (7-methylated or non-methylated) (7m-G-O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-adenosine cap (Appp), and any modified or unmodified nucleotide cap structure (N—O-5′-(HO)(O)P—O—(HO)(O)P—O—P(HO)(O)—O-5′), 5′-monothiophosphate (phosphorothioate; (HO)₂(S)P—O—5′), 5′-monodithiophosphate (phosphorodithioate; (HO)(HS)(S)P—O-5′), 5′-phosphorothiolate ((HO)₂(O)P—S-5′); any additional combination of oxgen/sulfur replaced monophosphate, diphosphate and triphosphates (e.g. 5′-alpha-thiotriphosphate, 5′-gamma-thiotriphosphate, etc.), 5′-phosphoramidates ((HO)₂(O)P—NH-5′, (HO)(NH₂)(O)P—O—5′), 5′-alkylphosphonates (R=alkyl=methyl, ethyl, isopropyl, propyl, etc., e.g. RP(OH)(O)—O-5′-, (OH)₂(O)P-5′-CH₂—), 5′-alkyletherphosphonates (R=alkylether=methoxymethyl (MeOCH₂—), ethoxymethyl, etc., e.g. RP(OH)(O)—O-5′-)).

A² is:

A³ is:

and

A⁴ is:

; H; Z⁴; an inverted nucleotide; an abasic nucleotide; or absent.

W¹ is OH, (CH₂)_(n)R¹⁰ , (CH₂)_(n)NHR¹⁰ , (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰; O(CH₂)_(n)R¹⁰; O(CH₂)_(n)OR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰; O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰, NH(CH₂)_(n)R¹⁰; NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰, S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)R¹⁰, S(CH₂)_(n)SR¹⁰O(CH₂CH₂O)_(m)CH₂CH₂OR¹⁰; O(CH₂CH₂O)_(m)CH₂CH₂NHR¹⁰, NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰, O-Q-R′° S-Q-R¹⁰ or —O—. W⁴ is O, CH₂, NH, or S.

X¹, X², X³, and X⁴ are each, independently, O or S.

Y¹, Y², Y³, and Y⁴ are each, independently, OH, O⁻, OR⁸, S, Se, BH₃ ⁻, H, NHR⁹, N(R⁹)₂ alkyl, cycloalkyl, aralkyl, aryl, or heteroaryl, each of which may be optionally substituted.

Z¹, Z², and Z³ are each independently O, CH₂, NH, or S. Z⁴ is OH, (CH₂)_(n)R¹⁰, (CH₂)_(n)NHR¹⁰, (CH₂)_(n)OR¹⁰, (CH₂)_(n)SR¹⁰; O(CH₂)_(n)R¹⁰; O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)NR¹⁰, O(CH₂)_(n)SR¹⁰, O(CH₂)_(n)SS(CH₂)_(n)OR¹⁰, O(CH₂)_(n)C(O)OR¹⁰; NH(CH₂)_(n)R¹⁰; NH(CH₂)_(n)NR¹⁰; NH(CH₂)_(n)OR¹⁰, NH(CH₂)_(n)SR¹⁰; S(CH₂)_(n)R¹⁰, S(CH₂)_(n)NR¹⁰, S(CH₂)_(n)OR¹⁰, S(CH₂)_(n)SR¹⁰ O(CH₂CH₂O)_(n)CH₂CH₂OR¹⁰, O(CH₂CH₂O)_(n)CH₂CH₂NHR¹⁰. NH(CH₂CH₂NH)_(m)CH₂CH₂NHR¹⁰; Q-R¹⁰; O-Q-R¹⁰N-Q-R¹⁰, S-Q-R¹⁰.

x is 5-100, chosen to comply with a length for an RNA agent described herein.

R⁷ is H; or is together combined with R⁴, R⁵, or R⁶ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid; and R¹⁰ is H; fluorophore (pyrene, TAMRA, fluorescein, Cy3 or Cy5 dyes); sulfur, silicon, boron or ester protecting group; intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4,texaphyrin,

Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipohilic carriers (cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino; alkyl, cycloalkyl, aryl, aralkyl, heteroaryl; radiolabelled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles); or an RNA agent. m is 0-1,000,000, and n is 0-20. Q is a spacer selected from the group consisting of abasic sugar, amide, carboxy, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, biotin or fluorescein reagents.

Preferred RNA agents in which the entire phosphate group has been replaced have the following structure (see Formula 3 below):

Referring to Formula 3, A¹⁰-A⁴⁰ is L-G-L; A¹⁰ and/or A⁴⁰ may be absent, in which L is a linker, wherein one or both L may be present or absent and is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g). G is a functional group selected from the group consisting of siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methyl enedimethylhydrazo and methyleneoxymethylimino.

R¹⁰, R²⁰, and R³⁰ are each, independently, H, (i.e. abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4-triazoles, 2-pyridinone, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thiocytosine, N6-methyl adenine, N6-isopentyladenine, 2-methylthio-N6-isopentenyladenine, N-methylguanines, or O-alkylated bases.

R⁴⁰, R⁵⁰, and R⁶⁰ are each, independently, OR⁸, O(CH₂CH₂O)_(m)CH₂CH₂OR⁸; O(CH₂)_(n)R⁹; O(CH₂)_(n)OR⁹, H; halo; NH₂; NHR⁸; N(R⁸)₂; NH(CH₂CH₂NH)_(m)CH₂CH₂R⁹; NHC(O)R⁸;; cyano; mercapto, SR⁷; alkyl-thio-alkyl; alkyl, aralkyl, cycloalkyl, aryl, heteroaryl, alkenyl, alkynyl, each of which may be optionally substituted with halo, hydroxy, oxo, nitro, haloalkyl, alkyl, alkaryl, aryl, aralkyl, alkoxy, aryloxy, amino, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, acylamino, alkylcarbamoyl, arylcarbamoyl, aminoalkyl, alkoxycarbonyl, carboxy, hydroxyalkyl, alkanesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano, and ureido groups; or R^(40,) R⁵⁰, or R⁶⁰ together combine with R⁷⁰ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

x is 5-100 or chosen to comply with a length for an RNA agent described herein.

R⁷⁰ is H; or is together combined with R⁴⁰, R⁵⁰or R⁶⁰ to form an [—O—CH₂—] covalently bound bridge between the sugar 2′ and 4′ carbons.

R⁸ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heteroaryl, amino acid, or sugar; and R⁹ is NH₂, alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid. m is 0-1,000,000, n is 0-20, and g is 0-2.

Preferred nucleoside surrogates have the following structure (see Formula 4 below):

SLR¹⁰⁰-(M-SLR²⁰⁰)_(x)-M-SLR³⁰⁰   FORMULA 4

S is a nucleoside surrogate selected from the group consisting of mophilino, cyclobutyl, pyrrolidine and peptide nucleic acid. L is a linker and is selected from the group consisting of CH₂(CH₂)_(g); N(CH₂)_(g); O(CH₂)_(g); S(CH₂)_(g); —C(O)(CH₂)_(n)— or may be absent. M is an amide bond; sulfonamide; sulfinate; phosphate group; modified phosphate group as described herein; or may be absent.

R¹⁰⁰, R²⁰⁰, and R³⁰⁰ are each, independently, H (i.e., abasic nucleotides), adenine, guanine, cytosine and uracil, inosine, thymine, xanthine, hypoxanthine, nubularine, tubercidine, isoguanisine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 5-halouracil, 5-(2-aminopropyl)uracil, 5-amino allyl uracil, 8-halo, amino, thiol, thioalkyl, hydroxyl and other 8-substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine, 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine, dihydrouracil, 3-deaza-5-azacytosine, 2-aminopurine, 5-alkyluracil, 7-alkylguanine, 5-alkyl cytosine,7-deazaadenine, 7-deazaguanine, N6, N6-dimethyladenine, 2,6-diaminopurine, 5-amino-allyl-uracil, N3-methyluracil substituted 1,2,4,-triazoles, 2-pyridinones, 5-nitroindole, 3-nitropyrrole, 5-methoxyuracil, uracil-5-oxyacetic acid, 5-methoxycarbonylmethyluracil, 5-methyl-2-thiouracil, 5-methoxycarbonylmethyl-2-thiouracil, 5-methylaminomethyl-2-thiouracil, 3-(3-amino-3carboxypropyl)uracil, 3-methylcytosine, 5-methylcytosine, N⁴-acetyl cytosine, 2-thioicytosine, N6-methyladenine, N6-isopentyl adenine, 2-methylthio-N6-isopentenyl adenine, N-methylguanines, or O-alkylated bases.

x is 5-100, or chosen to comply with a length for an RNA agent described herein; and g is 0-2.

Nuclease Resistant Monomers

In one aspect, the invention features a nuclease resistant monomer, or a an iRNA agent which incorporates a nuclease resistant monomer (NMR), such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/469,612 (Attorney Docket No. 14174-069P01), filed on May 9, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a NMR and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a NMR.

An iRNA agent can include monomers which have been modifed so as to inhibit degradation, e.g., by nucleases, e.g., endonucleases or exonucleases, found in the body of a subject. These monomers are referred to herein as NRM's, or nuclease resistance promoting monomers or modifications. In many cases these modifications will modulate other properties of the iRNA agent as well, e.g., the ability to interact with a protein, e.g., a transport protein, e.g., serum albumin, or a member of the RISC (RNA-induced Silencing Complex), or the ability of the first and second sequences to form a duplex with one another or to form a duplex with another sequence, e.g., a target molecule.

While not wishing to be bound by theory, it is believed that modifications of the sugar, base, and/or phosphate backbone in an iRNA agent can enhance endonuclease and exonuclease resistance, and can enhance interactions with transporter proteins and one or more of the functional components of the RISC complex. Preferred modifications are those that increase exonuclease and endonuclease resistance and thus prolong the halflife of the iRNA agent prior to interaction with the RISC complex, but at the same time do not render the iRNA agent resistant to endonuclease activity in the RISC complex. Again, while not wishing to be bound by any theory, it is believed that placement of the modifications at or near the 3′ and/or 5′ end of antisense strands can result in iRNA agents that meet the preferred nuclease resistance criteria delineated above. Again, still while not wishing to be bound by any theory, it is believed that placement of the modifications at e.g., the middle of a sense strand can result in iRNA agents that are relatively less likely to undergo off-targeting.

Modifications described herein can be incorporated into any double-standed RNA and RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the modifications described herein. The anti sense strand may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. The sense strand may include modifications at the 3′ end and/or the 5′ end and/or at any one of the intervening positions between the two ends of the strand. The iRNA agent may also include a duplex comprising two hybridized antisense strands. The first and/or the second antisense strand may include one or more of the modifications described herein. Thus, one and/or both antisense strands may include modifications at the 3′ end and/or the 5′ end and/or at one or more positions that occur 1-6 (e.g., 1-5, 1-4, 1-3, 1-2) nucleotides from either end of the strand. Particular configurations are discussed below.

Modifications that can be useful for producing iRNA agents that meet the preferred nuclease resistance criteria delineated above can include one or more of the following chemical and/or stereochemical modifications of the sugar, base, and/or phosphate backbone:

(i) chiral (Sp) thioates. Thus, preferred NRM's include nucleotide dimers with an enriched or pure for a particular chiral form of a modified phosphate group containing a heteroatom at the nonbridging position, e.g., S_(P) or Rp, at the position X, where this is the position normally occupied by the oxygen. The atom at X can also be S, Se, Nr₂, or Br₃. When X is S, enriched or chirally pure S_(P) linkage is preferred. Enriched means at least 70, 80, 90, 95, or 99% of the preferred form. Such NRM's are discussed in more detail below;

(ii) attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. Thus, preferred NRM's include monomers at the terminal position derivitized at a cationic group. As the 5′ end of an antisense sequence should have a terminal —OH or phosphate group this NRM is preferraly not used at th 5′ end of an anti-sense sequence. The group should be attached at a position on the base which minimizes intererence with H bond formation and hybridization, e.g., away form the face which intereacts with the complementary base on the other strand, e.g, at the 5′ position of a pyrimidine or a 7-position of a purine. These are discussed in more detail below;

(iii) nonphosphate linkages at the termini. Thus, preferred NRM's include Non-phosphate linkages, e.g., a linkage of 4 atoms which confers greater resistance to cleavage than does a phosphate bond. Examples include 3′ CH2-NCH₃-O—CH2-5′ and 3′ CH2-NH—(O═)—CH2-5′.;

(iv) 3′-bridging thiophosphates and 5′-bridging thiophosphates. Thus, preferred NRM's can inlcuded these structures;

(v) L-RNA, 2′-5′ likages, inverted linkages, a-nucleosides. Thus, other preferred NRM's include: L nucleosides and dimeric nucleotides derived from L-nucleosides; 2′-5′ phosphate, non-phosphate and modified phosphate linkages (e.g., thiophospahtes, phosphoramidates and boronophosphates); dimers having inverted linkages, e.g., 3′-3′ or 5′-5′ linkages; monomers having an alpha linkage at the 1′ site on the sugar, e.g., the structures described herein having an alpha linkage;

(vi) conjugate groups. Thus, preferred NRM's can include e.g., a targeting moiety or a conjugated ligand described herein conjugated with the monomer, e.g., through the sugar, base, or backbone;

(vi) abasic linkages. Thus, preferred NRM's can include an abasic monomer, e.g., an abasic monomer as described herein (e.g., a nucleobaseless monomer); an aromatic or heterocyclic or polyheterocyclic aromatic monomer as described herein.; and

(vii) 5′-phosphonates and 5′-phosphate prodrugs. Thus, preferred NRM's include monomers, preferably at the terminal position, e.g., the 5′ position, in which one or more atoms of the phosphate group is derivatized with a protecting group, which protecting group or groups, are removed as a result of the action of a component in the subject's body, e.g, a carboxyesterase or an enzyme present in the subject's body. E.g., a phosphate prodrug in which a carboxy esterase cleaves the protected molecule resulting in the production of a thioate anion which attacks a carbon adjacent to the O of a phosphate and resulting in the production of an uprotected phosphate.

One or more different NRM modifications can be introduced into an iRNA agent or into a sequence of an iRNA agent. An NRM modification can be used more than once in a sequence or in an iRNA agent. As some NRM's interfere with hybridization the total number incorporated, should be such that acceptable levels of iRNA agent duplex formation are maintainted.

In some embodiments NRM modifications are introduced into the terminal the cleavage site or in the cleavage region of a sequence (a sense strand or sequence) which does not target a desired sequence or gene in the subject. This can reduce off-target silencing.

Chiral S_(P) Thioates

A modification can include the alteration, e.g., replacement, of one or both of the non-linking (X and Y) phosphate oxygens and/or of one or more of the linking (W and Z) phosphate oxygens. Formula X below depicts a phosphate moiety linking two sugar/sugar surrogate-base moities, SB₁ and SB₂.

In certain embodiments, one of the non-linking phosphate oxygens in the phosphate backbone moiety (X and Y) can be replaced by any one of the following: S, Se, BR₃ (R is hydrogen, alkyl, aryl, etc.), C (i.e., an alkyl group, an aryl group, etc.), H, NR₂ (R is hydrogen, alkyl, aryl, etc.), or OR (R is alkyl or aryl). The phosphorus atom in an unmodified phosphate group is achiral. However, replacement of one of the non-linking oxygens with one of the above atoms or groups of atoms renders the phosphorus atom chiral; in other words a phosphorus atom in a phosphate group modified in this way is a stereogenic center. The stereogenic phosphorus atom can possess either the “R” configuration (herein R_(P)) or the “S” configuration (herein S_(P)). Thus if 60% of a population of stereogenic phosphorus atoms have the R_(P) configuration, then the remaining 40% of the population of stereogenic phosphorus atoms have the S_(P) configuration.

In some embodiments, iRNA agents, having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms, may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the S_(P) configuration. Alternatively, iRNA agents having phosphate groups in which a phosphate non-linking oxygen has been replaced by another atom or group of atoms may contain a population of stereogenic phosphorus atoms in which at least about 50% of these atoms (e.g., at least about 60% of these atoms, at least about 70% of these atoms, at least about 80% of these atoms, at least about 90% of these atoms, at least about 95% of these atoms, at least about 98% of these atoms, at least about 99% of these atoms) have the R_(P) configuration. In other embodiments, the population of stereogenic phosphorus atoms may have the S_(P) configuration and may be substantially free of stereogenic phosphorus atoms having the R_(P) configuration. In still other embodiments, the population of stereogenic phosphorus atoms may have the R_(P) configuration and may be substantially free of stereogenic phosphorus atoms having the S_(P) configuration. As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the R_(P) configuration” means that moieties containing stereogenic phosphorus atoms having the R_(P) configuration cannot be detected by conventional methods known in the art (chiral HPLC, ¹H NMR analysis using chiral shift reagents, etc.). As used herein, the phrase “substantially free of stereogenic phosphorus atoms having the S_(P) configuration” means that moieties containing stereogenic phosphorus atoms having the S_(P) configuration cannot be detected by conventional methods known in the art (chiral HPLC, ¹H NMR analysis using chiral shift reagents, etc.).

In a preferred embodiment, modified iRNA agents contain a phosphorothioate group, i.e., a phosphate groups in which a phosphate non-linking oxygen has been replaced by a sulfur atom. In an especially preferred embodiment, the population of phosphorothioate stereogenic phosphorus atoms may have the S_(P) configuration and be substantially free of stereogenic phosphorus atoms having the R_(P) configuration.

Phosphorothioates may be incorporated into iRNA agents using dimers e.g., formulas X-1 and X-2. The former can be used to introduce phosphorothioate

at the 3′ end of a strand, while the latter can be used to introduce this modification at the 5′ end or at a position that occurs e.g., 1, 2, 3, 4, 5, or 6 nucleotides from either end of the strand. In the above formulas, Y can be 2-cyanoethoxy, W and Z can be O, R₂ can be, e.g., a substituent that can impart the C-3 endo configuration to the sugar (e.g., OH, F, OCH₃), DMT is dimethoxytrityl, and “BASE” can be a natural, unusual, or a universal base.

X-1 and X-2 can be prepared using chiral reagents or directing groups that can result in phosphorothioate-containing dimers having a population of stereogenic phosphorus atoms having essentially only the R_(P) configuration (i.e., being substantially free of the Sp configuration) or only the S_(P) configuration (i.e., being substantially free of the Rp configuration). Alternatively, dimers can be prepared having a population of stereogenic phosphorus atoms in which about 50% of the atoms have the R_(P) configuration and about 50% of the atoms have the S_(P) configuration. Dimers having stereogenic phosphorus atoms with the R_(P) configuration can be identified and separated from dimers having stereogenic phosphorus atoms with the S_(P) configuration using e.g., enzymatic degradation and/or conventional chromatography techniques.

Cationic Groups

Modifications can also include attachment of one or more cationic groups to the sugar, base, and/or the phosphorus atom of a phosphate or modified phosphate backbone moiety. A cationic group can be attached to any atom capable of substitution on a natural, unusual or universal base. A preferred position is one that does not interfere with hybridization, i.e., does not interfere with the hydrogen bonding interactions needed for base pairing. A cationic group can be attached e.g., through the C2′ position of a sugar or analogous position in a cyclic or acyclic sugar surrogate. Cationic groups can include e.g., protonated amino groups, derived from e.g., O-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); aminoalkoxy, e.g., O(CH₂)_(n)AMINE, (e.g., AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, or diheteroaryl amino, ethylene diamine, polyamino); amino (e.g. NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino, diheteroaryl amino, or amino acid); or NH(CH₂CH₂NH)_(n)CH₂CH₂-AMINE (AMINE=NH₂; alkylamino, dialkylamino, heterocyclyl, arylamino, diaryl amino, heteroaryl amino,or diheteroaryl amino).

Nonphosphate Linkages

Modifications can also include the incorporation of nonphosphate linkages at the 5′ and/or 3′ end of a strand. Examples of nonphosphate linkages which can replace the phosphate group include methyl phosphonate, hydroxylamino, siloxane, carbonate, carboxymethyl, carbamate, amide, thioether, ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, oxime, methyleneimino, methylenemethylimino, methylenehydrazo, methyl enedimethyl hydrazo and methyleneoxymethylimino. Preferred replacements include the methyl phosphonate and hydroxylamino groups.

3′-Bridging Thiophosphates and 5′-bridging Thiophosphates; Locked-RNA, 2′-5′ Likages, Inverted Linkages, α-nucleosides; Conjugate Groups; Abasic Linkages; and 5′-phosphonates and 5′-Phosphate Prodrugs

Referring to formula X above, modifications can include replacement of one of the bridging or linking phosphate oxygens in the phosphate backbone moiety (W and Z). Unlike the situation where only one of X or Y is altered, the phosphorus center in the phosphorodithioates is achiral which precludes the formation of iRNA agents containing a stereogenic phosphorus atom.

Modifications can also include linking two sugars via a phosphate or modified phosphate group through the 2′ position of a first sugar and the 5′ position of a second sugar. Also contemplated are inverted linkages in which both a first and second sugar are eached linked through the respective3′ positions. Modified RNA's can also include “abasic” sugars, which lack a nucleobase at C-1′. The sugar group can also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified iRNA agent can include nucleotides containing e.g., arabinose, as the sugar. In another subset of this modification, the natural, unusual, or universal base may have the α-configuration. Modifcations can also include L-RNA.

Modifications can also include 5′-phosphonates, e.g., P(O)(O⁻)₂—X—C^(5′)-sugar (X═CH2, CF2, CHF and 5′-phosphate prodrugs, e.g., P(O)[OCH2CH2SC(O)R]₂CH₂C⁵′-sugar. In the latter case, the prodrug groups may be decomposed via reaction first with carboxy esterases. The remaining ethyl thiolate group via intramolecular S_(N)2 displacement can depart as episulfide to afford the underivatized phosphate group.

Modification can also include the addition of conjugating groups described elseqhere herein, which are prefereably attached to an iRNA agent through any amino group available for conjugation.

Nuclease resistant modifications include some which can be placed only at the terminus and others which can go at any position. Generally the modifications that can inhibit hybridization so it is preferably to use them only in terminal regions, and preferrable to not use them at the cleavage site or in the cleavage region of an sequence which targets a subject sequence or gene. The can be used anywhere in a sense sequence, provided that sufficient hybridization between the two sequences of the iRNA agent is maintained. In some embodiments it is desirabable to put the NRM at the cleavage site or in the cleavage region of a sequence which does not target a subject sequence or gene,as it can minimize off-target silencing.

In addition, an iRNA agent described herein can have an overhang which does not form a duplex structure with the other sequence of the iRNA agent—it is an overhang, but it does hybridize, either with itself, or with another nucleic acid, other than the other sequence of the iRNA agent.

In most cases, the nuclease-resistance promoting modifications will be distributed differently depending on whether the sequence will target a sequence in the subject (often referred to as an anti-sense sequence) or will not target a sequence in the subject (often referred to as a sense sequence). If a sequence is to target a sequence in the subject, modifications which interfer with or inhibit endonuclease cleavage should not be inserted in the region which is subject to RISC mediated cleavage, e.g., the cleavage site or the cleavage region (As described in Elbashir et al., 2001, Genes and Dev. 15: 188, hereby incorporated by reference, cleavage of the target occurs about in the middle of a 20 or 21 nt guide RNA, or about 10 or 11 nucleotides upstream of the first nucleotide which is complementary to the guide sequence. As used herein cleavage site refers to the nucleotide on either side of the cleavage site, on the target or on the iRNA agent strand which hybridizes to it. Cleavage region means an nucleotide with 1, 2, or 3 nucletides of the cleave site, in either direction.)

Such modifications can be introduced into the terminal regions, e.g., at the terminal position or with 2, 3, 4, or 5 positions of the terminus, of a sequence which targets or a sequence which does not target a sequence in the subject.

An iRNA agent can have a first and a second strand chosen from the following:

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region;

a first strand which does not target a sequence and which has an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end; and

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which does not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end(5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).

An iRNA agent can also target two sequences and can have a first and second strand chosen from:

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

a first strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a first strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;

a first strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end(5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand) and

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end;

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end (5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand);

a second strand which targets a sequence and which has an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end and which has a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end;

a second strand which targets a sequence and which preferably does not have an an NRM modification at the cleavage site or in the cleavage region;

a second strand which targets a sequence and which dose not have an NRM modification at the cleavage site or in the cleavage region and one or more of an NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 3′ end, a NRM modification at or within 1, 2, 3, 4, 5, or 6 positions from the 5′ end, or NRM modifications at or within 1, 2, 3, 4, 5, or 6 positions from both the 3′ and the 5′ end(5′ end NRM modifications are preferentially not at the terminus but rather at a position 1, 2, 3, 4, 5, or 6 away from the 5′ terminus of an antisense strand).

Ribose Mimics

In one aspect, the invention features a ribose mimic, or an iRNA agent which incorporates a ribose mimic, such as those described herein and those described in copending co-owned U.S. Provisional Application Ser. No. 60/454,962 (Attorney Docket No. 14174-064P01), filed on Mar. 13, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a ribose mimic and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a ribose mimic.

Thus, an aspect of the invention features an iRNA agent that includes a secondary hydroxyl group, which can increase efficacy and/or confer nuclease resistance to the agent. Nucleases, e.g., cellular nucleases, can hydrolyze nucleic acid phosphodiester bonds, resulting in partial or complete degradation of the nucleic acid. The secondary hydroxy group confers nuclease resistance to an iRNA agent by rendering the iRNA agent less prone to nuclease degradation relative to an iRNA which lacks the modification. While not wishing to be bound by theory, it is believed that the presence of a secondary hydroxyl group on the iRNA agent can act as a structural mimic of a 3′ ribose hydroxyl group, thereby causing it to be less susceptible to degradation.

The secondary hydroxyl group refers to an “OH” radical that is attached to a carbon atom substituted by two other carbons and a hydrogen. The secondary hydroxyl group that confers nuclease resistance as described above can be part of any acyclic carbon-containing group. The hydroxyl may also be part of any cyclic carbon-containing group, and preferably one or more of the following conditions is met (1) there is no ribose moiety between the hydroxyl group and the terminal phosphate group or (2) the hydroxyl group is not on a sugar moiety which is coupled to a base.. The hydroxyl group is located at least two bonds (e.g., at least three bonds away, at least four bonds away, at least five bonds away, at least six bonds away, at least seven bonds away, at least eight bonds away, at least nine bonds away, at least ten bonds away, etc.) from the terminal phosphate group phosphorus of the iRNA agent. In preferred embodiments, there are five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group.

Preferred iRNA agent delivery modules with five intervening bonds between the terminal phosphate group phosphorus and the secondary hydroxyl group have the following structure (see formula Y below):

Referring to formula Y, A is an iRNA agent, including any iRNA agent described herein. The iRNA agent may be connected directly or indirectly (e.g., through a spacer or linker) to “W” of the phosphate group. These spacers or linkers can include e.g., —(CH₂)_(n)—, —(CH₂)_(n)N—, —(CH₂)_(n)O—, —(CH₂)_(n)S—, O(CH₂CH₂O)_(n)CH₂CH₂OH (e.g., n=3 or 6), abasic sugars, amide, carboxy, amine, oxyamine, oxyimine, thioether, disulfide, thiourea, sulfonamide, or morpholino, or biotin and fluorescein reagents.

The iRNA agents can have a terminal phosphate group that is unmodified (e.g., W, X, Y, and Z are O) or modified. In a modified phosphate group, W and Z can be independently NH, O, or S; and X and Y can be independently S, Se, BH₃ ⁻, C₁-C₆ alkyl, C₆-C₁₀ aryl, H, O, O⁻, alkoxy or amino (including alkylamino, arylamino, etc.). Preferably, W, X and Z are O and Y is S.

R₁ and R₃ are each, independently, hydrogen; or C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl.

R₂ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₂ may be taken together with with R₄ or R₆ to form a ring of 5-12 atoms.

R₄ is hydrogen; C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₄ may be taken together with with R₂ or R₅ to form a ring of 5-12 atoms.

R₅ is hydrogen, C₁-C₁₀₀ alkyl optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl; or, when n is 1, R₅ may be taken together with with R₄ to form a ring of 5-12 atoms.

R₆ is hydrogen, C₁-C₁₀₀ alkyl, optionally substituted with hydroxyl, amino, halo, phosphate or sulfate and/or may be optionally inserted with N, O, S, alkenyl or alkynyl, or, when n is 1, R₆ may be taken together with with R₂ to form a ring of 6-10 atoms;

R₇ is hydrogen, C₁-C₁₀₀ alkyl, or C(O)(CH₂)_(q)C(O)NHR₉; T is hydrogen or a functional group; n and q are each independently 1-100; R_(g) is C₁-C₁₀ alkyl or C₆-C₁₀ aryl; and R₉ is hydrogen, C₁-C₁₀ alkyl, C₆-C₁₀ aryl or a solid support agent.

Preferred embodiments may include one of more of the following subsets of iRNA agent delivery modules.

In one subset of RNAi agent delivery modules, A can be connected directly or indirectly through a terminal 3′ or 5′ ribose sugar carbon of the RNA agent.

In another subset of RNAi agent delivery modules, X, W, and Z are O and Y is S.

In still yet another subset of RNAi agent delivery modules, n is 1, and R₂ and R₆ are taken together to form a ring containing six atoms and R₄ and R₅ are taken together to form a ring containing six atoms. Preferably, the ring system is a trans-decalin. For example, the RNAi agent delivery module of this subset can include a compound of Formula (Y-1):

The functional group can be, for example, a targeting group (e.g., a steroid or a carbohydrate), a reporter group (e.g., a fluorophore), or a label (an isotopically labelled moiety). The targeting group can further include protein binding agents, endothelial cell targeting groups (e.g., RGD peptides and mimetics), cancer cell targeting groups (e.g., folate Vitamin B12, Biotin), bone cell targeting groups (e.g., bisphosphonates, polyglutamates, polyaspartates), multivalent mannose (for e.g., macrophage testing), lactose, galactose, N-acetyl-galactosamine, monoclonal antibodies, glycoproteins, lectins, melanotropin, or thyrotropin.

As can be appreciated by the skilled artisan, methods of synthesizing the compounds of the formulae herein will be evident to those of ordinary skill in the art. The synthesized compounds can be separated from a reaction mixture and further purified by a method such as column chromatography, high pressure liquid chromatography, or recrystallization. Additionally, the various synthetic steps may be performed in an alternate sequence or order to give the desired compounds. Synthetic chemistry transformations and protecting group methodologies (protection and deprotection) useful in synthesizing the compounds described herein are known in the art and include, for example, those such as described in R. Larock, Comprehensive Organic Transformations, VCH Publishers (1989); T. W. Greene and P. G. M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents for Organic Synthesis, John Wiley and Sons (1994); and L. Paquette, ed., Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons (1995), and subsequent editions thereof.

Ribose Replacement Monomer Subunits

iRNA agents can be modified in a number of ways which can optimize one or more characteristics of the iRNA agent. In one aspect, the invention features a ribose replacement monomer subunit (RRMS), or a an iRNA agent which incorporates a RRMS, such as those described herein and those described in one or more of U.S. Provisional Application Ser. No. 60/493,986 (Attorney Docket No. 14174-079P01), filed on Aug. 8, 2003, which is hereby incorporated by reference; U.S. Provisional Application Ser. No. 60/494,597 (Attorney Docket No. 14174-080P01), filed on Aug. 11, 2003, which is hereby incorporated by reference; U.S. Provisional Application Ser. No. 60/506,341 (Attorney Docket No. 14174-080P02), filed on Sep. 26, 2003, which is hereby incorporated by reference; and in U.S. Provisional Application Ser. No. 60/158,453 (Attorney Docket No. 14174-080P03), filed on Nov. 7, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a RRMS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an archtecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a RRMS.

The ribose sugar of one or more ribonucleotide subunits of an iRNA agent can be replaced with another moiety, e.g., a non-carbohydrate (preferably cyclic) carrier. A ribonucleotide subunit in which the ribose sugar of the subunit has been so replaced is referred to herein as a ribose replacement modification subunit (RRMS). A cyclic carrier may be a carbocyclic ring system, i.e., all ring atoms are carbon atoms, or a heterocyclic ring system, i.e., one or more ring atoms may be a heteroatom, e.g., nitrogen, oxygen, sulfur. The cyclic carrier may be a monocyclic ring system, or may contain two or more rings, e.g. fused rings. The cyclic carrier may be a fully saturated ring system, or it may contain one or more double bonds.

The carriers further include (i) at least two “backbone attachment points” and (ii) at least one “tethering attachment point.” A “backbone attachment point” as used herein refers to a functional group, e.g. a hydroxyl group, or generally, a bond available for, and that is suitable for incorporation of the carrier into the backbone, e.g., the phosphate, or modified phosphate, e.g., sulfur containing, backbone, of a ribonucleic acid. A “tethering attachment point” as used herein refers to a constituent ring atom of the cyclic carrier, e.g., a carbon atom or a heteroatom (distinct from an atom which provides a backbone attachment point), that connects a selected moiety. The moiety can be, e.g., a ligand, e.g., a targeting or delivery moiety, or a moiety which alters a physical property, e.g., lipophilicity, of an iRNA agent. Optionally, the selected moiety is connected by an intervening tether to the cyclic carrier. Thus, it will include a functional group, e.g., an amino group, or generally, provide a bond, that is suitable for incorporation or tethering of another chemical entity, e.g., a ligand to the constituent ring.

Incorporation of one or more RRMSs described herein into an RNA agent, e.g., an iRNA agent, particularly when tethered to an appropriate entity, can confer one or more new properties to the RNA agent and/or alter, enhance or modulate one or more existing properties in the RNA molecule. E.g., it can alter one or more of lipophilicity or nuclease resistance. Incorporation of one or more RRMSs described herein into an iRNA agent can, particularly when the RRMS is tethered to an appropriate entity, modulate, e.g., increase, binding affinity of an iRNA agent to a target mRNA, change the geometry of the duplex form of the iRNA agent, alter distribution or target the iRNA agent to a particular part of the body, or modify the interaction with nucleic acid binding proteins (e.g., during RISC formation and strand separation).

Accordingly, in one aspect, the invention features, an iRNA agent preferably comprising a first strand and a second strand, wherein at least one subunit having a formula (R-1) is incorporated into at least one of said strands.

Referring to formula (R-1), X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁸, O, S, CR⁹R¹⁰, or absent; and Z is CR¹¹R¹² or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a), OR^(b), (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b), provided that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is OR^(a) or OR^(b) and that at least one of R¹, R², R³, R⁴, R⁹, and R¹⁰ is (CH₂)_(n)OR^(a), or (CH₂)_(n)OR^(b) (when the RRMS is terminal, one of R¹, R², R³, R⁴, R⁹, and R¹⁰ will include R^(a) and one will include R^(b); when the RRMS is internal, two of R¹, R², R³, R⁴, R⁹, and R¹⁰ will each include an R^(b)); further provided that preferably OR^(a) may only be present with (CH₂)_(n)OR^(b) and (CH₂)_(n)OR^(a) may only be present with OR^(b).

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, H, C₁-C₆ alkyl optionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ together are C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is C₁-C₂₀ alkyl substituted with NR^(c)R^(d); R⁸ is C₁-C₆ alkyl; R¹³ is hydroxy, C₁-C₄ alkoxy, or halo; and R¹⁴ is NR^(c)R⁷.

R^(a) is:

and

R^(b) is:

Each of A and C is, independently, O or S.

B is OH, O⁻, or

R^(c) is H or C1-C6 alkyl; R^(d) is H or a ligand; and n is 1-4.

In a preferred embodiment the ribose is replaced with a pyrroline scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is absent.

In other preferred embodiments the ribose is replaced with a piperidine scaffold, and X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a piperazine scaffold, and X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a morpholino scaffold, and X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹².

In other preferred embodiments the ribose is replaced with a decalin scaffold, and X isCH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and together are C⁶ cycloalkyl.

In other preferred embodiments the ribose is replaced with a decalin/indane scafold and , and X is CH₂; Y is CR⁹R¹⁰; and Z is CR¹¹R¹²; and R⁵ and R¹¹ together are C⁵ cycloalkyl.

In other preferred embodiments, the ribose is replaced with a hydroxyproline scaffold.

RRMSs described herein may be incorporated into any double-stranded RNA-like molecule described herein, e.g., an iRNA agent. An iRNA agent may include a duplex comprising a hybridized sense and antisense strand, in which the antisense strand and/or the sense strand may include one or more of the RRMSs described herein. An RRMS can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be internal, and will preferably be positioned in regions not critical for antisense binding to the target.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and at (or within 1, 2, or 3 positions of) the 3′ end of the sense strand. In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both ligands are located at the same end of the iRNA agent.

In certain embodiments, two ligands are tethered, preferably, one on each strand and are hydrophobic moieties. While not wishing to be bound by theory, it is believed that pairing of the hydrophobic ligands can stabilize the iRNA agent via intermolecular van der Waals interactions.

In an embodiment, an iRNA agent may have an RRMS at (or within 1, 2, or 3 positions of) the 3′ end of the antisense strand and an RRMS at the 5′ end of the sense strand, in which both RRMSs may share the same ligand (e.g., cholic acid) via connection of their individual tethers to separate positions on the ligand. A ligand shared between two proximal RRMSs is referred to herein as a “hairpin ligand.”

In other embodiments, an iRNA agent may have an RRMS at the 3′ end of the sense strand and an RRMS at an internal position of the sense strand. An iRNA agent may have an RRMS at an internal position of the sense strand; or may have an RRMS at an internal position of the antisense strand; or may have an RRMS at an internal position of the sense strand and an RRMS at an internal position of the antisense strand.

In preferred embodiments the iRNA agent includes a first and second sequences, which are preferably two separate molecules as opposed to two sequences located on the same strand, have sufficient complementarity to each other to hybridize (and thereby form a duplex region), e.g., under physiological conditions, e.g., under physiological conditions but not in contact with a helicase or other unwinding enzyme.

It is preferred that the first and second sequences be chosen such that the ds iRNA agent includes a single strand or unpaired region at one or both ends of the molecule. Thus, a ds iRNA agent contains first and second sequences, preferable paired to contain an overhang, e.g., one or two 5′ or 3′ overhangs but preferably a 3′ overhang of 2-3 nucleotides. Most embodiments will have a 3′ overhang. Preferred sRNA agents will have single-stranded overhangs, preferably 3′ overhangs, of 1 or preferably 2 or 3 nucleotides in length at each end. The overhangs can be the result of one strand being longer than the other, or the result of two strands of the same length being staggered. 5′ ends are preferably phosphorylated.

An RNA agent, e.g., an iRNA agent, containing a preferred, but nonlimiting RRMS is presented as formula (R-2) in FIG. 4. The carrier includes two “backbone attachment points” (hydroxyl groups), a “tethering attachment point,” and a ligand, which is connected indirectly to the carrier via an intervening tether. The RRMS may be the 5′ or 3′ terminal subunit of the RNA molecule, i.e., one of the two “W” groups may be a hydroxyl group, and the other “W” group may be a chain of two or more unmodified or modified ribonucleotides. Alternatively, the RRMS may occupy an internal position, and both “W” groups may be one or more unmodified or modified ribonucleotides. More than one RRMS may be present in a RNA molecule, e.g., an iRNA agent.

The modified RNA molecule of formula (R-2) can be obtained using oligonucleotide synthetic methods known in the art. In a preferred embodiment, the modified RNA molecule of formula (II) can be prepared by incorporating one or more of the corresponding RRMS monomer compounds (RRMS monomers, see, e.g., A, B, and C in FIG. 4) into a growing sense or antisense strand, utilizing, e.g., phosphoramidite or H-phosphonate coupling strategies.

The RRMS monomers generally include two differently functionalized hydroxyl groups (OFG¹ and OFG² above), which are linked to the carrier molecule (see A in FIG. 4), and a tethering attachment point. As used herein, the term “functionalized hydroxyl group” means that the hydroxyl proton has been replaced by another substituent. As shown in representative structures B and C, one hydroxyl group (OFG') on the carrier is functionalized with a protecting group (PG). The other hydroxyl group (OFG²) can be functionalized with either (1) a liquid or solid phase synthesis support reagent (solid circle) directly or indirectly through a linker, L, as in B, or (2) a phosphorus-containing moiety, e.g., a phosphoramidite as in C. The tethering attachment point may be connected to a hydrogen atom, a tether, or a tethered ligand at the time that the monomer is incorporated into the growing sense or antisense strand (see R in Scheme 1). Thus, the tethered ligand can be, but need not be attached to the monomer at the time that the monomer is incorporated into the growing strand. In certain embodiments, the tether, the ligand or the tethered ligand may be linked to a “precursor” RRMS after a “precursor” RRMS monomer has been incorporated into the strand.

The (OFG¹) protecting group may be selected as desired, e.g., from T.W. Greene and P.G.M. Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley and Sons (1991). The protecting group is preferably stable under amidite synthesis conditions, storage conditions, and oligonucleotide synthesis conditions. Hydroxyl groups, —OH, are nucleophilic groups (i.e., Lewis bases), which react through the oxygen with electrophiles (i.e., Lewis acids). Hydroxyl groups in which the hydrogen has been replaced with a protecting group, e.g., a triarylmethyl group or a trialkylsilyl group, are essentially unreactive as nucleophiles in displacement reactions. Thus, the protected hydroxyl group is useful in preventing e.g., homocoupling of compounds exemplified by structure C during oligonucleotide synthesis. A preferred protecting group is the dimethoxytrityl group.

When the OFG² in B includes a linker, e.g., a long organic linker, connected to a soluble or insoluble support reagent, solution or solid phase synthesis techniques can be employed to build up a chain of natural and/or modified ribonucleotides once OFG¹ is deprotected and free to react as a nucleophile with another nucleoside or monomer containing an electrophilic group (e.g., an amidite group). Alternatively, a natural or modified ribonucleotide or oligoribonucleotide chain can be coupled to monomer C via an amidite group or H-phosphonate group at OFG². Subsequent to this operation, OFG¹ can be deblocked, and the restored nucleophilic hydroxyl group can react with another nucleoside or monomer containing an electrophilic group (see FIG. 1). R′ can be substituted or unsubstituted alkyl or alkenyl. In preferred embodiments, R′ is methyl, allyl or 2-cyanoethyl. R″ may a C₁-C₁₀ alkyl group, preferably it is a branched group containing three or more carbons, e.g., isopropyl.

OFG² in B can be hydroxyl functionalized with a linker, which in turn contains a liquid or solid phase synthesis support reagent at the other linker terminus. The support reagent can be any support medium that can support the monomers described herein. The monomer can be attached to an insoluble support via a linker, L, which allows the monomer (and the growing chain) to be solubilized in the solvent in which the support is placed. The solubilized, yet immobilized, monomer can react with reagents in the surrounding solvent; unreacted reagents and soluble by-products can be readily washed away from the solid support to which the monomer or monomer-derived products is attached. Alternatively, the monomer can be attached to a soluble support moiety, e.g., polyethylene glycol (PEG) and liquid phase synthesis techniques can be used to build up the chain. Linker and support medium selection is within skill of the art. Generally the linker may be —C(O)(CH₂)_(q)C(O)—, or —C(O)(CH₂)_(q)S—, preferably, it is oxalyl, succinyl or thioglycolyl. Standard control pore glass solid phase synthesis supports can not be used in conjunction with fluoride labile 5′ silyl protecting groups because the glass is degraded by fluoride with a significant reduction in the amount of full-length product. Fluoride-stable polystyrene based supports or PEG are preferred.

Preferred carriers have the general formula (R-3) provided below. (In that structure preferred backbone attachment points can be chosen from R¹ or R²; R³ or R⁴; or R⁹ and R¹⁰ if Y is CR⁹R¹⁰ (two positions are chosen to give two backbone attachment points, e.g., R¹ and R⁴, or R⁴ and R⁹. Preferred tethering attachment points include R⁷; R⁵ or R⁶ when X is CH₂. The carriers are described below as an entity, which can be incorporated into a strand. Thus, it is understood that the structures also encompass the situations wherein one (in the case of a terminal position) or two (in the case of an internal position) of the attachment points, e.g., le or R²; R³ or R⁴; or R⁹ or R¹⁰ (when Y is CR⁹R¹⁰), is connected to the phosphate, or modified phosphate, e.g., sulfur containing, backbone. E.g., one of the above-named R groups can be —CH2-, wherein one bond is connected to the carrier and one to a backbone atom, e.g., a linking oxygen or a central phosphorus atom.)

X is N(CO)R⁷, NR⁷ or CH₂; Y is NR⁹, O, S, CR⁹R¹⁰; and Z is CR¹¹R¹² or absent.

Each of R¹, R², R³, R⁴, R⁹, and R¹⁰ is, independently, H, OR^(a), or (CH₂)_(n)OR^(b), provided that at least two of R¹, R², R³, R⁴, R⁹, and R¹⁰ are OR^(a) and/or (CH₂)_(n)OR^(b).

Each of R⁵, R⁶, R¹¹, and R¹² is, independently, a ligand, H, C₁-C₆ alkyl optionally substituted with 1-3 R¹³, or C(O)NHR⁷; or R⁵ and R¹¹ together are C₃-C₈ cycloalkyl optionally substituted with R¹⁴.

R⁷ is H, a ligand, or C₁-C₂₀ alkyl substituted with NR^(c)R^(d); R⁸ is H or C₁-C₆ alkyl; R¹³ is hydroxy, C₁-C₄ alkoxy, or halo; R¹⁴ is NR^(c)R⁷; R¹⁵ is C₁-C₆ alkyl optionally substituted with cyano, or C₂-C₆ alkenyl; R¹⁶ is C₁-C₁₀ alkyl; and R¹⁷ is a liquid or solid phase support reagent.

L is —C(O)(CH₂)_(q)C(O)—, or —C(O)(CH₂)_(q)S—; R^(a) is CAr₃; R^(b) is P(O)(O⁻)H, P(OR¹⁵)N(R¹⁶)₂or L-R¹⁷; R^(c) is H or C₁-C₆ alkyl; and R^(d) is H or a ligand.

Each Ar is, independently, C₆-C₁₀ aryl optionally substituted with C₁-C₄ alkoxy; n is 1-4; and q is 0-4.

Exemplary carriers include those in which, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰ , and Z is absent; or X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰ , and Z is CR¹¹R¹², or X is N(CO)R⁷ or NR⁷, Y is NR⁸, and Z is CR¹¹R¹², or X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹², or X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and R¹¹ together form C₅ cycloalkyl (H, z=1).

In certain embodiments, the carrier may be based on the pyrroline ring system or the 3-hydroxyproline ring system, e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰ , and Z is absent (D). OFG¹ is preferably attached to a primary carbon, e.g., an exocyclic alkylene

group, e.g., a methylene group, connected to one of the carbons in the five-membered ring (—CH₂OFG¹ in D). OFG² is preferably attached directly to one of the carbons in the five-membered ring (—OFG² in D). For the pyrroline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3; or —CH₂OFG¹ may be attached to C-3 and OFG² may be attached to C-4. In certain embodiments, CH₂OFG¹ and OFG² may be geminally substituted to one of the above-referenced carbons.For the 3-hydroxyproline-based carriers, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-4. The pyrroline- and 3-hydroxyproline-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperidine ring system (E), e.g., X is N(CO)R⁷ or NR⁷, Y is CR⁹R¹⁰ , and Z is CR¹¹R¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group (n=1) or ethylene group (n=2), connected to one of the carbons in the six-membered ring [—(CH₂)_(n)OFG¹ in E]. OFG² is preferably attached directly to one of the carbons in the six-membered ring (—OFG² in E). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, or C-4. Alternatively, —(CH₂)_(n)OFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ may be attached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-4; or -(CH₂)_(n)OFG¹ may be attached to C-4 and OFG² may be attached to C-3. The piperidine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

In certain embodiments, the carrier may be based on the piperazine ring system (F), e.g., X is N(CO)R⁷ or NR⁷, Y is N⁸, and Z is CR¹¹R¹², or the morpholine ring system (G), e.g., X is N(CO)R⁷ or NR⁷, Y is O, and Z is CR¹¹R¹². OFG¹ is preferably

attached to a primary carbon, e.g., an exocyclic alkylene group, e.g., a methylene group, connected to one of the carbons in the six-membered ring (—CH₂OFG¹ in F or G). OFG² is preferably attached directly to one of the carbons in the six-membered rings (—OFG² in F or G). For both F and G, —CH₂OFG¹ may be attached to C-2 and OFG² may be attached to C-3;

or vice versa. In certain embodiments, CH₂OFG¹ and OFG² may be geminally substituted to one of the above-referenced carbons.The piperazine- and morpholine-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, CH₂OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. R′″ can be, e.g., C₁-C₆ alkyl, preferably CH₃. The tethering attachment point is preferably nitrogen in both F and G.

In certain embodiments, the carrier may be based on the decalin ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹² , and R⁵ and R¹¹ together form C₆ cycloalkyl (H, z=2), or the indane ring system, e.g., X is CH₂; Y is CR⁹R¹⁰; Z is CR¹¹R¹², and R⁵ and together form C₅ cycloalkyl (H, z=1). OFG¹ is preferably attached to a primary carbon,

e.g., an exocyclic methylene group (n=1) or ethylene group (n=2) connected to one of C-2, C-3, C-4, or C-5 [—(CH₂)_(n)OFG¹ in H]. OFG² is preferably attached directly to one of C-2, C-3, C-4, or C-5 (—OFG² in H). —(CH₂)_(n)OFG¹ and OFG² may be disposed in a geminal manner on the ring, i.e., both groups may be attached to the same carbon, e.g., at C-2, C-3, C-4, or C-5. Alternatively, —(CH₂)_(n)OFG¹ and OFG² may be disposed in a vicinal manner on the ring, i.e., both groups may be attached to adjacent ring carbon atoms, e.g., —(CH₂)_(n)OFG¹ may be attached to C-2 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-2; —(CH₂)_(n)OFG¹ may be attached to C-3 and OFG² may be attached to C-4; or —(CH₂)_(n)OFG¹ may be attached to C-4 and OFG² may be attached to C-3; —(CH₂)_(n)OFG¹ may be attached to C-4 and OFG² may be attached to C-5; or -(CH₂)_(n)OFG¹ may be attached to C-5 and OFG² may be attached to C-4. The decalin or indane-based monomers may therefore contain linkages (e.g., carbon-carbon bonds) wherein bond rotation is restricted about that particular linkage, e.g. restriction resulting from the presence of a ring. Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another in any of the pairings delineated above. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. In a preferred embodiment, the substituents at C-1 and C-6 are trans with respect to one another. The tethering attachment point is preferably C-6 or C-7.

Other carriers may include those based on 3-hydroxyproline (J). Thus, —(CH₂)_(n)OFG¹ and OFG² may be cis or trans with respect to one another. Accordingly, all cis/trans isomers are expressly included. The monomers may also contain one or more asymmetric centers

and thus occur as racemates and racemic mixtures, single enantiomers, individual diastereomers and diastereomeric mixtures. All such isomeric forms of the monomers are expressly included. The tethering attachment point is preferably nitrogen.

Representative carriers are shown in FIG. 5.

In certain embodiments, a moiety, e.g., a ligand may be connected indirectly to the carrier via the intermediacy of an intervening tether. Tethers are connected to the carrier at the tethering attachment point (TAP) and may include any C₁-C₁₀₀ carbon-containing moiety, (e.g. C₁-C₇₅, C₁-C₅₀, C₁-C₂₀, C₁-C₁₀, C₁-C₆), preferably having at least one nitrogen atom. In preferred embodiments, the nitrogen atom forms part of a terminal amino group on the tether, which may serve as a connection point for the ligand. Preferred tethers (underlined) include TAP-(CH₂)₂NH₂ ; TAP-C(O)(CH₂NH₂ ; or TAP-NR′″″(CH₂)₂NH₂ , in which n is 1-6 and R′″″ is C₁-C₆ alkyl, and R^(d) is hydrogen or a ligand. In other embodiments, the nitrogen may form part of a terminal oxyamino group, e.g., —ONH₂, or hydrazino group, —NHNH₂. The tether may optionally be substituted, e.g., with hydroxy, alkoxy, perhaloalkyl, and/or optionally inserted with one or more additional heteroatoms, e.g., N, O, or S. Preferred tethered ligands may include, e.g., TAP-(CH₂)_(n)NH(LIGAND), TAP-C(O)(CH₂)_(n)NH(LIGAND), or TAP-NR′″″(CH₂)_(n)NH(LIGAND); TAP-(CH₂)₂ONH(LIGAND), TAP-C(O)(CH₂)_(n)ONH(LIGAND), or TAP-NR′″″(CH₂)_(n)ONH(LIGAND); TAP-(CH₂)_(n)NHNH₂(LIGAND), TAP-C(O)(CH₂)_(n)NHNH₂(LIGAND), or TAP-NR′″″(CH₂)_(n)NHNH₂(LIGAND).

In other embodiments the tether may include an electrophilic moiety, preferably at the terminal position of the tether. Preferred electrophilic moieties include, e.g., an aldehyde, alkyl halide, mesylate, tosylate, nosylate, or brosylate, or an activated carboxylic acid ester, e.g. an NHS ester, or a pentafluorophenyl ester. Preferred tethers (underlined) include TAP-(CH₂)_(n)CHO; TAP-C(O)(CH₂)_(n)CHO; or TAP-NR″″(CH₂)_(n)CHO, in which n is 1-6 and R″″ is C₁-C₆ alkyl; or TAP-(CH₂)_(n)C(O)ONHS; TAP-C(O)(CH₂)_(n)ONHS; or TAP-NR″″(CH₂)_(n)C(O)ONHS, in which n is 1-6 and R″″ is C₁-C₆ alkyl; TAP-(CH₂)_(n)C(O)OC₆F₅ ; TAP-C(O)(CH₂)_(n)C(O)OC₆F₅ ; or TAP-NR″″(CH₂)_(n)C(O)OC₆F₅ , in which n is 1-6 and R″″ is C₁-C₆ alkyl; or —(CH₂)_(n)CH₂LG; TAP-C(O)(CH₂)_(n)CH₂LG; or TAP-NR″″(CH₂)_(n)CH₂LG, in which n is 1-6 and R″″ is C₁-C₆ alkyl (LG can be a leaving group, e.g., halide, mesylate, tosylate, nosylate, brosylate). Tethering can be carried out by coupling a nucleophilic group of a ligand, e.g., a thiol or amino group with an electrophilic group on the tether.

Tethered Entities

A wide variety of entities can be tethered to an iRNA agent, e.g., to the carrier of an RRMS. Examples are described below in the context of an RRMS but that is only preferred, entities can be coupled at other points to an iRNA agent.

Preferred moieties are ligands, which are coupled, preferably covalently, either directly or indirectly via an intervening tether, to the RRMS carrier. In preferred embodiments, the ligand is attached to the carrier via an intervening tether. As discussed above, the ligand or tethered ligand may be present on the RRMS monomer when the RRMS monomer is incorporated into the growing strand. In some embodiments, the ligand may be incorporated into a “precursor” RRMS after a “precursor” RRMS monomer has been incorporated into the growing strand. For example, an RRMS monomer having, e.g., an amino-terminated tether (i.e., having no associated ligand), e.g., TAP-(CH₂)_(n)NH₂ may be incorporated into a growing sense or antisense strand. In a subsequent operation, i.e., after incorporation of the precursor monomer into the strand, a ligand having an electrophilic group, e.g., a pentafluorophenyl ester or aldehyde group, can subsequently be attached to the precursor RRMS by coupling the electrophilic group of the ligand with the terminal nucleophilic group of the precursor RRMS tether.

In preferred embodiments, a ligand alters the distribution, targeting or lifetime of an iRNA agent into which it is incorporated. In preferred embodiments a ligand provides an enhanced affinity for a selected target, e.g, molecule, cell or cell type, compartment, e.g., a cellular or organ compartment, tissue, organ or region of the body, as, e.g., compared to a species absent such a ligand. Preferred ligands will not take part in duplex pairing in a duplexed nucleic acid.

Preferred ligands can improve transport, hybridization, and specificity properties and may also improve nuclease resistance of the resultant natural or modified oligoribonucleotide, or a polymeric molecule comprising any combination of monomers described herein and/or natural or modified ribonucleotides.

Ligands in general can include therapeutic modifiers, e.g., for enhancing uptake; diagnostic compounds or reporter groups e.g., for monitoring distribution; cross-linking agents; and nuclease-resistance conferring moieties. General examples include lipids, steroids, vitamins, sugars, proteins, peptides, polyamines, and peptide mimics.

Ligands can include a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand may also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polyamino acid is a polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Example of polyamines include: polyethylenimine, polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide or RGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g. acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA), lipophilic molecules, e.g, cholesterol, cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine)and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin), transport/absorption facilitators (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules having a specific affinity for a co-ligand, or antibodies e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, or bone cell. Ligands may also include hormones and hormone receptors. They can also include non-peptidic species, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. The ligand can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g, a drug, which can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The ligand can increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary ligands that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

In one aspect, the ligand is a lipid or lipid-based molecule. Such a lipid or lipid-based molecule preferably binds a serum protein, e.g., human serum albumin (HSA). An HSA binding ligand allows for distribution of the conjugate to a target tissue, e.g., a non-kidney target tissue of the body. Preferably, the target tissue is the liver, preferably parenchymal cells of the liver. Other molecules that can bind HSA can also be used as ligands. For example, neproxin or aspirin can be used. A lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport into a target cell or cell membrane, and/or (c) can be used to adjust binding to a seru protein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the binding of the conjugate to a target tissue. For example, a lipid or lipid-based ligand that binds to HSA more strongly will be less likely to be targeted to the kidney and therefore less likely to be cleared from the body. A lipid or lipid-based ligand that binds to HSA less strongly can be used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably, it binds HSA with a sufficient affinity such that the conjugate will be preferably distributed to a non-kidney tissue. However, it is preferred that the affinity not be so strong that the HSA-ligand binding cannot be reversed.

In another preferred embodiment, the lipid based ligand binds HSA weakly or not at all, such that the conjugate will be preferably distributed to the kidney. Other moieties that target to kidney cells can also be used in place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which is taken up by a target cell, e.g., a proliferating cell. These are particularly useful for treating disorders characterized by unwanted cell proliferation, e.g., of the malignant or non-malignant type, e.g., cancer cells. Exemplary vitamins include vitamin A, E, and K. Other exemplary vitamins include are B vitamin, e.g., folic acid, B12, riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up by cancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably a helical cell-permeation agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide such as that or antennopedia. If the agent is a peptide, it can be modified, including a peptidylmimetic, invertomers, non-peptide or pseudo-peptide linkages, and use of D-amino acids. The helical agent is preferably an alpha-helical agent, which preferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (also referred to herein as an oligopeptidomimetic) is a molecule capable of folding into a defined three-dimensional structure similar to a natural peptide. The attachment of peptide and peptidomimetics to iRNA agents can affect pharmacokinetic distribution of the iRNA, such as by enhancing cellular recognition and absorption. The peptide or peptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long (see Table 1, for example).

TABLE 1 Exemplary Cell Permeation Peptides Cell Permeation Peptide Amino acid Sequence Reference Penetratin RQIKIWFQNRRMKWKK (SEQ ID NO: 6737) Derossi etal., J. Biol. Chem. 269:10444, 1994 Tat fragment GRKKRRQRRRPPQC (SEQ ID NO: 6738) Vives etal., J. Biol. (48-60) Chem., 272:16010, 1997 Signal GALFLGWLGAAGSTMGAWSQPKKKRKV Chaloin etal., Sequence- (SEQ ID NO: 6738) Biochem. Biophys. based peptide Res. Commun., 243:601, 1998 PVEC LLIILRRRIRKQAHAHSK (SEQ ID NO: 6739) Elmquist etal., Exp. Cell Res., 269:237, 2001 Transportan GWTLNSAGYLLKINLKALAALAKKIL Pooga etal., FASEB (SEQ ID NO: 6740) J., 12:67, 1998 Amphiphilic KLALKLALKALKAALKLA (SEQ ID Oehlke etal., Mol. model peptide NO: 6741) Ther., 2:339, 2000 Arg₉ RRRRRRRRR (SEQ ID NO: 6742) Mitchell etal., J. Pept. Res., 56:318, 2000 Bacterial cell KFFKFFKFFK (SEQ ID NO: 6743) wall permeating LL-37 LLGDFFRKSKEKIGKEFKRIVQRIKDFLRN LVPRTES (SEQ ID NO: 6744) Cecropin P1 SWLSKTAKKLENSAKKRISEGIAIAIQGGP R (SEQ ID NO: 6745) α-defensin ACYCRIPACIAGERRYGTCIYQGRLWAFC C (SEQ ID NO: 6746) b-defensin DHYNCVSSGGQCLYSACPIFTKIQGTCYR GKAKCCK (SEQ ID NO: 6747) Bactenecin RKCRIVVIRVCR (SEQ ID NO: 6748) PR-39 RRRPRPPYLPRPRPPPFFPPRLPPRIPPGFPP RFPPRFPGKR-NH2 (SEQ ID NO: 6749) Indolicidin ILPWKWPWWPWRR-NH2 (SEQ ID NO: 6750)

A peptide or peptidomimetic can be, for example, a cell permeation peptide, cationic peptide, amphipathic peptide, or hydrophobic peptide (e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety can be a dendrimer peptide, constrained peptide or crosslinked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO:6751). An RFGF analogue (e.g., amino acid sequence AALLPVLLAAP (SEQ ID NO:6752)) containing a hydrophobic MTS can also be a targeting moiety. The peptide moiety can be a “delivery” peptide, which can carry large polar molecules including peptides, oligonucleotides, and protein across cell membranes. For example, sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:6753)) and the Drosophila Antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO:6754)) have been found to be capable of functioning as delivery peptides. A peptide or peptidomimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage-display library, or one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to an iRNA agent via an incorporated monomer unit is a cell targeting peptide such as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. A peptide moiety can range in length from about 5 amino acids to about 40 amino acids. The peptide moieties can have a structural modification, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as an endothelial tumor cell or a breast cancer tumor cell (Zitzmann et al., Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targeting of an iRNA agent to tumors of a variety of other tissues, including the lung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy 8:783-787, 2001). The RGD peptide can be linear or cyclic, and can be modified, e.g., glycosylated or methylated to facilitate targeting to specific tissues. For example, a glycosylated RGD peptide can deliver an iRNA agent to a tumor cell expressing a_(v)B₃ (Haubner et al., Jour. Nucl. Med., 42:326-336, 2001).

Peptides that target markers enriched in proliferating cells can be used. E.g., RGD containing peptides and peptidomimetics can target cancer cells, in particular cells that exhibit an I_(v)Θ₃ integrin. Thus, one could use RGD peptides, cyclic peptides containing RGD, RGD peptides that include D-amino acids, as well as synthetic RGD mimics. In addition to RGD, one can use other moieties that target the I_(v)-Θ₃ integrin ligand. Generally, such ligands can be used to control proliferating cells and angiogeneis. Preferred conjugates of this type include an iRNA agent that targets PECAM-1, VEGF, or other cancer gene, e.g., a cancer gene described herein.

A “cell permeation peptide” is capable of permeating a cell, e.g., a microbial cell, such as a bacterial or fungal cell, or a mammalian cell, such as a human cell. A microbial cell-permeating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin or bactenecin), or a peptide containing only one or two dominating amino acids (e.g., PR-39 or indolicidin). A cell permeation peptide can also include a nuclear localization signal (NLS). For example, a cell permeation peptide can be a bipartite amphipathic peptide, such as MPG, which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res. 31:2717-2724, 2003).

In one embodiment, a targeting peptide tethered to an RRMS can be an amphipathic a-helical peptide. Exemplary amphipathic a-helical peptides include, but are not limited to, cecropins, lycotoxins, paradaxins, buforin, CPF, bombinin-like peptide (BLP), cathelicidins, ceratotoxins, S. clava peptides, hagfish intestinal antimicrobial peptides (HFIAPs), magainines, brevinins-2, dermaseptins, melittins, pleurocidin, H₂A peptides, Xenopus peptides, esculentinis-1, and caerins. A number of factors will preferably be considered to maintain the integrity of helix stability. For example, a maximum number of helix stabilization residues will be utilized (e.g., leu, ala, or lys), and a minimum number helix destabilization residues will be utilized (e.g., proline, or cyclic monomeric units. The capping residue will be considered (for example Gly is an exemplary N-capping residue and/or C-terminal amidation can be used to provide an extra H-bond to stabilize the helix. Formation of salt bridges between residues with opposite charges, separated by i±3, or i±4 positions can provide stability. For example, cationic residues such as lysine, arginine, homo-arginine, ornithine or histidine can form salt bridges with the anionic residues glutamate or aspartate.

Peptide and petidomimetic ligands include those having naturally occurring or modified peptides, e.g., D or L peptides; α, β, or γ peptides; N-methyl peptides; azapeptides; peptides having one or more amide, i.e., peptide, linkages replaced with one or more urea, thiourea, carbamate, or sulfonyl urea linkages; or cyclic peptides.

Methods for Making iRNA Agents

iRNA agents can include modified or non-naturally occuring bases, e.g., bases described in copending and coowned U.S. Provisional Application Ser. No. 60/463,772 (Attorney Docket No. 14174-070P01), filed on Apr. 17, 2003, which is hereby incorporated by reference and/or in copending and coowned U.S. Provisional Application Ser. No. 60/465,802 (Attorney Docket No. 14174-074P01), filed on Apr. 25, 2003, which is hereby incorporated by reference. Monomers and iRNA agents which include such bases can be made by the methods found in U.S. Provisional Application Ser. No. 60/463,772 (Attorney Docket No. 14174-070P01), filed on Apr. 17, 2003, and/or in U.S. Provisional Application Ser. No. 60/465,802 (Attorney Docket No. 14174-074P01), filed on Apr. 25, 2003.

In addition, the invention includes iRNA agents having a modified or non-naturally occuring base and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a modified or non-naturally occuring base.

The synthesis and purification of oligonucleotide peptide conjugates can be performed by established methods. See, for example, Trufert et al., Tetrahedron, 52:3005, 1996; and Manoharan, “Oligonucleotide Conjugates in Antisense Technology,” in Antisense Drug Technology, ed. S.T. Crooke, Marcel Dekker, Inc., 2001.

In one embodiment of the invention, a peptidomimetic can be modified to create a constrained peptide that adopts a distinct and specific preferred conformation, which can increase the potency and selectivity of the peptide. For example, the constrained peptide can be an azapeptide (Gante, Synthesis, 405-413, 1989). An azapeptide is synthesized by replacing the a-carbon of an amino acid with a nitrogen atom without changing the structure of the amino acid side chain. For example, the azapeptide can be synthesized by using hydrazine in traditional peptide synthesis coupling methods, such as by reacting hydrazine with a “carbonyl donor,” e.g., phenylchloroformate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an N-methyl peptide. N-methyl peptides are composed of N-methyl amino acids, which provide an additional methyl group in the peptide backbone, thereby potentially providing additional means of resistance to proteolytic cleavage. N-methyl peptides can by synthesized by methods known in the art (see, for example, Lindgren et al., Trends Pharmacol. Sci. 21:99, 2000; Cell Penetrating Peptides: Processes and Applications, Langel, ed., CRC Press, Boca Raton, Fla., 2002; Fische et al., Bioconjugate. Chem. 12: 825, 2001; Wander et al., J. Am. Chem. Soc., 124:13382, 2002). For example, an Ant or Tat peptide can be an N-methyl peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a β-peptide. β-peptides form stable secondary structures such as helices, pleated sheets, turns and hairpins in solutions. Their cyclic derivatives can fold into nanotubes in the solid state. β-peptides are resistant to degradation by proteolytic enzymes. β-peptides can be synthesized by methods known in the art. For example, an Ant or Tat peptide can be a β-peptide.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be a oligocarbamate. Oligocarbamate peptides are internalized into a cell by a transport pathway facilitated by carbamate transporters. For example, an Ant or Tat peptide can be an oligocarbamate.

In one embodiment of the invention, a peptide or peptidomimetic (e.g., a peptide or peptidomimetic tethered to an RRMS) can be an oligourea conjugate (or an oligothiourea conjugate), in which the amide bond of a peptidomimetic is replaced with a urea moiety. Replacement of the amide bond provides increased resistance to degradation by proteolytic enzymes, e.g., proteolytic enzymes in the gastrointestinal tract. In one embodiment, an oligourea conjugate is tethered to an iRNA agent for use in oral delivery. The backbone in each repeating unit of an oligourea peptidomimetic can be extended by one carbon atom in comparison with the natural amino acid. The single carbon atom extension can increase peptide stability and lipophilicity, for example. An oligourea peptide can therefore be advantageous when an iRNA agent is directed for passage through a bacterial cell wall, or when an iRNA agent must traverse the blood-brain barrier, such as for the treatment of a neurological disorder. In one embodiment, a hydrogen bonding unit is conjugated to the oligourea peptide, such as to create an increased affinity with a receptor. For example, an Ant or Tat peptide can be an oligourea conjugate (or an oligothiourea conjugate).

The siRNA peptide conjugates of the invention can be affiliated with, e.g., tethered to, RRMSs occurring at various positions on an iRNA agent. For example, a peptide can be terminally conjugated, on either the sense or the antisense strand, or a peptide can be bisconjugated (one peptide tethered to each end, one conjugated to the sense strand, and one conjugated to the antisense strand). In another option, the peptide can be internally conjugated, such as in the loop of a short hairpin iRNA agent. In yet another option, the peptide can be affiliated with a complex, such as a peptide-carrier complex.

A peptide-carrier complex consists of at least a carrier molecule, which can encapsulate one or more iRNA agents (such as for delivery to a biological system and/or a cell), and a peptide moiety tethered to the outside of the carrier molecule, such as for targeting the carrier complex to a particular tissue or cell type. A carrier complex can carry additional targeting molecules on the exterior of the complex, or fusogenic agents to aid in cell delivery. The one or more iRNA agents encapsulated within the carrier can be conjugated to lipophilic molecules, which can aid in the delivery of the agents to the interior of the carrier.

A carrier molecule or structure can be, for example, a micelle, a liposome (e.g., a cationic liposome), a nanoparticle, a microsphere, or a biodegradable polymer. A peptide moiety can be tethered to the carrier molecule by a variety of linkages, such as a disulfide linkage, an acid labile linkage, a peptide-based linkage, an oxyamino linkage or a hydrazine linkage. For example, a peptide-based linkage can be a GFLG peptide. Certain linkages will have particular advantages, and the advantages (or disadvantages) can be considered depending on the tissue target or intended use. For example, peptide based linkages are stable in the blood stream but are susceptible to enzymatic cleavage in the lysosomes.

Targeting

The iRNA agents of the invention are particularly useful when targeted to the liver. An iRNA agent can be targeted to the liver by incorporation of an RRMS containing a ligand that targets the liver. For example, a liver-targeting agent can be a lipophilic moiety. Preferred lipophilic moieties include lipid, cholesterols, oleyl, retinyl, or cholesteryl residues. Other lipophilic moieties that can function as liver-targeting agents include cholic acid, adamantane acetic acid, 1-pyrene butyric acid, dihydrotestosterone, 1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid, myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid, dimethoxytrityl, or phenoxazine.

An iRNA agent can also be targeted to the liver by association with a low-density lipoprotein (LDL), such as lactosylated LDL. Polymeric carriers complexed with sugar residues can also function to target iRNA agents to the liver.

A targeting agent that incorporates a sugar, e.g., galactose and/or analogues thereof, is particularly useful. These agents target, in particular, the parenchymal cells of the liver. For example, a targeting moiety can include more than one or preferably two or three galactose moieties, spaced about 15 angstroms from each other. The targeting moiety can alternatively be lactose (e.g., three lactose moieties), which is glucose coupled to a galactose. The targeting moiety can also be N-Acetyl-Galactosamine, N-Ac-Glucosamine. A mannose or mannose-6-phosphate targeting moiety can be used for macrophage targeting.

Conjugation of an iRNA agent with a serum albumin (SA), such as human serum albumin, can also be used to target the iRNA agent to the liver.

An iRNA agent targeted to the liver by an RRMS targeting moiety described herein can target a gene expressed in the liver. For example, the iRNA agent can target p21(WAF1/DIP1), P27(KIP1), the a-fetoprotein gene, beta-catenin, or c-MET, such as for treating a cancer of the liver. In another embodiment, the iRNA agent can target apoB-100, such as for the treatment of an HDL/LDL cholesterol imbalance; dyslipidemias, e.g., familial combined hyperlipidemia (FCHL), or acquired hyperlipidemia; hypercholesterolemia; statin-resistant hypercholesterolemia; coronary artery disease (CAD); coronary heart disease (CHD); or atherosclerosis. In another embodiment, the iRNA agent can target forkhead homologue in rhabdomyosarcoma (FKHR); glucagon; glucagon receptor; glycogen phosphorylase; PPAR-Gamma Coactivator (PGC-1); Fructose-1,6-bisphosphatase; glucose-6-phosphatase; glucose-6-phosphate translocator; glucokinase inhibitory regulatory protein; or phosphoenolpyruvate carboxykinase (PEPCK), such as to inhibit hepatic glucose production in a mammal, such as a human, such as for the treatment of diabetes. In another embodiment, an iRNA agent targeted to the liver can target Factor V, e.g., the Leiden Factor V allele, such as to reduce the tendency to form a blood clot. An iRNA agent targeted to the liver can include a sequence which targets hepatitis virus (e.g., Hepatitis A, B, C, D, E, F, G, or H). For example, an iRNA agent of the invention can target any one of the nonstructural proteins of HCV: NS3, 4A, 4B, 5A, or 5B. For the treatment of hepatitis B, an iRNA agent can target the protein X (HBx) gene, for example.

Preferred ligands on RRMSs include folic acid, glucose, cholesterol, cholic acid, Vitamin E, Vitamin K, or Vitamin A.

Definitions

The term “halo” refers to any radical of fluorine, chlorine, bromine or iodine.

The term “alkyl” refers to a hydrocarbon chain that may be a straight chain or branched chain, containing the indicated number of carbon atoms. For example, C₁-C₁₂ alkyl indicates that the group may have from 1 to 12 (inclusive) carbon atoms in it. The term “haloalkyl” refers to an alkyl in which one or more hydrogen atoms are replaced by halo, and includes alkyl moieties in which all hydrogens have been replaced by halo (e.g., perfluoroalkyl). Alkyl and haloalkyl groups may be optionally inserted with O, N, or S. The terms “aralkyl” refers to an alkyl moiety in which an alkyl hydrogen atom is replaced by an aryl group. Aralkyl includes groups in which more than one hydrogen atom has been replaced by an aryl group. Examples of “aralkyl” include benzyl, 9-fluorenyl, benzhydryl, and trityl groups.

The term “alkenyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more double bonds. Examples of a typical alkenyl include, but not limited to, allyl, propenyl, 2-butenyl, 3-hexenyl and 3-octenyl groups. The term “alkynyl” refers to a straight or branched hydrocarbon chain containing 2-8 carbon atoms and characterized in having one or more triple bonds. Some examples of a typical alkynyl are ethynyl, 2-propynyl, and 3-methylbutynyl, and propargyl. The sp² and sp^(a) carbons may optionally serve as the point of attachment of the alkenyl and alkynyl groups, respectively.

The term “alkoxy” refers to an -O-alkyl radical. The term “aminoalkyl” refers to an alkyl substituted with an aminoThe term “mercapto” refers to an —SH radical. The term “thioalkoxy” refers to an -S-alkyl radical.

The term “alkylene” refers to a divalent alkyl (i.e., —R—), e.g., —CH₂—, —CH₂CH₂—, and —CH₂CH₂CH₂—. The term “alkylenedioxo” refers to a divalent species of the structure —O—R—O—, in which R represents an alkylene.

The term “aryl” refers to an aromatic monocyclic, bicyclic, or tricyclic hydrocarbon ring system, wherein any ring atom capable of substitution can be substituted by a substituent. Examples of aryl moieties include, but are not limited to, phenyl, naphthyl, and anthracenyl.

The term “cycloalkyl” as employed herein includes saturated cyclic, bicyclic, tricyclic,or polycyclic hydrocarbon groups having 3 to 12 carbons, wherein any ring atom capable of substitution can be substituted by a substituent. The cycloalkyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of cycloalkyl moieties include, but are not limited to, cyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent. The heterocyclyl groups herein described may also contain fused rings. Fused rings are rings that share a common carbon-carbon bond. Examples of heterocyclyl include, but are not limited to tetrahydrofuranyl, tetrahydropyranyl, piperidinyl, morpholino, pyrrolinyl and pyrrolidinyl.

The term “heteroaryl” refers to an aromatic 5-8 membered monocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ring system having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms if bicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selected from O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms of N, O, or S if monocyclic, bicyclic, or tricyclic, respectively), wherein any ring atom capable of substitution can be substituted by a substituent.

The term “oxo” refers to an oxygen atom, which forms a carbonyl when attached to carbon, an N-oxide when attached to nitrogen, and a sulfoxide or sulfone when attached to sulfur.

The term “acyl” refers to an alkylcarbonyl, cycloalkylcarbonyl, arylcarbonyl, heterocyclylcarbonyl, or heteroarylcarbonyl substituent, any of which may be further substituted by sub stituents.

The term “substituents” refers to a group “substituted” on an alkyl, cycloalkyl, alkenyl, alkynyl, heterocyclyl, heterocycloalkenyl, cycloalkenyl, aryl, or heteroaryl group at any atom of that group. Suitable substituents include, without limitation, alkyl, alkenyl, alkynyl, alkoxy, halo, hydroxy, cyano, nitro, amino, SO₃H, sulfate, phosphate, perfluoroalkyl, perfluoroalkoxy, methylenedioxy, ethylenedioxy, carboxyl, oxo, thioxo, imino (alkyl, aryl, aralkyl), S(O)_(n)alkyl (where n is 0-2), S(O)_(n)aryl (where n is 0-2), S(O)_(n)heteroaryl (where n is 0-2), S(O)_(n) heterocyclyl (where n is 0-2), amine (mono-, di-, alkyl, cycloalkyl, aralkyl, heteroaralkyl, and combinations thereof), ester (alkyl, aralkyl, heteroaralkyl), amide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), sulfonamide (mono-, di-, alkyl, aralkyl, heteroaralkyl, and combinations thereof), unsubstituted aryl, unsubstituted heteroaryl, unsubstituted heterocyclyl, and unsubstituted cycloalkyl. In one aspect, the substituents on a group are independently any one single, or any subset of the aforementioned sub stituents.

The terms “adeninyl, cytosinyl, guaninyl, thyminyl, and uracilyl” and the like refer to radicals of adenine, cytosine, guanine, thymine, and uracil.

As used herein, an “unusual” nucleobase can include any one of the following:

2-methyladeninyl,

N6-methyladeninyl,

2-methylthio-N6-methyladeninyl,

N6-isopentenyladeninyl,

2-methylthio-N6-isopentenyladeninyl,

N6-(cis-hydroxyisopentenyl)adeninyl,

2-methylthio-N6-(cis-hydroxyisopentenyl) adeninyl,

N6-glycinylcarbamoyladeninyl,

N6-threonylcarbamoyladeninyl,

2-methylthio-N6-threonyl carbamoyladeninyl,

N6-methyl-N6-threonyl carbamoyladeninyl,

N6-hydroxynorvalylcarbamoyladeninyl,

2-methylthio-N6-hydroxynorvalyl carbamoyladeninyl,

N6,N6-dimethyladeninyl,

3-methylcytosinyl,

5-methylcytosinyl,

2-thiocytosinyl,

5-formylcytosinyl,

N4-methylcytosinyl,

5-hydroxymethylcytosinyl,

1-methylguaninyl,

N2-methylguaninyl,

7-methylguaninyl,

N2,N2-dimethylguaninyl,

N2,N2,7-trimethylguaninyl,

1-methylguaninyl,

7-cyano-7-deazaguaninyl,

7-aminomethyl-7-deazaguaninyl,

pseudouracilyl,

dihydrouracilyl,

5-methyluracilyl,

1-methylpseudouracilyl,

2-thiouracilyl,

4-thiouracilyl,

2-thiothyminyl

5-methyl-2-thiouracilyl,

3-(3-amino-3-carboxypropyl)uracilyl,

5-hydroxyuracilyl,

5-methoxyuracilyl,

uracilyl 5-oxyacetic acid,

uracilyl 5-oxyacetic acid methyl ester,

5-(carboxyhydroxymethyl)uracilyl,

5-(carboxyhydroxymethyl)uracilyl methyl ester,

5-methoxycarbonylmethyluracilyl,

5-methoxycarbonylmethyl-2-thiouracilyl,

5-aminomethyl-2-thiouracilyl,

5-methylaminomethyluracilyl,

5-methylaminomethyl-2-thiouracilyl,

5-methylaminomethyl-2-selenouracilyl,

5 -carb amoylmethyluracilyl,

5-carboxymethyl aminomethyluracilyl,

5-carboxymethyl aminomethyl-2-thiouracilyl,

3-methyluracilyl,

1-methyl-3 -(3 -amino-3 -carboxypropyl) pseudouracilyl,

5-carb oxym ethyluracilyl,

5-methyldihydrouracilyl, or

3-methylpseudouracilyl.

Asymmetrical Modifications

In one aspect, the invention features an iRNA agent which can be asymmetrically modified as described herein.

In addition, the invention includes iRNA agents having asymmetrical modifications and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates an asymmetrical modification.

iRNA agents of the invention can be asymmetrically modified. An asymmetrically modified iRNA agent is one in which a strand has a modification which is not present on the other strand. An asymmetrical modification is a modification found on one strand but not on the other strand. Any modification, e.g., any modification described herein, can be present as an asymmetrical modification. An asymmetrical modification can confer any of the desired properties associated with a modification, e.g., those properties discussed herein. E.g., an asymmetrical modification can: confer resistance to degradation, an alteration in half life; target the iRNA agent to a particular target, e.g., to a particular tissue; modulate, e.g., increase or decrease, the affinity of a strand for its complement or target sequence; or hinder or promote modification of a terminal moiety, e.g., modification by a kinase or other enzymes involved in the RISC mechanism pathway. The designation of a modification as having one property does not mean that it has no other property, e.g., a modification referred to as one which promotes stabilization might also enhance targeting.

While not wishing to be bound by theory or any particular mechanistic model, it is believed that asymmetrical modification allows an iRNA agent to be optimized in view of the different or “asymmetrical” functions of the sense and antisense strands. For example, both strands can be modified to increase nuclease resistance, however, since some changes can inhibit RISC activity, these changes can be chosen for the sense stand . In addition, since some modifications, e.g., targeting moieties, can add large bulky groups that, e.g., can interfere with the cleavage activity of the RISC complex, such modifications are preferably placed on the sense strand. Thus, targeting moieties, especially bulky ones (e.g. cholesterol), are preferentially added to the sense strand. In one embodiment, an asymmetrical modification in which a phosphate of the backbone is substituted with S, e.g., a phosphorothioate modification, is present in the antisense strand, and a 2′ modification, e.g., 2′ OMe is present in the sense strand. A targeting moiety can be present at either (or both) the 5′ or 3′ end of the sense strand of the iRNA agent. In a preferred example, a P of the backbone is replaced with S in the antisense strand, 2′OMe is present in the sense strand, and a targeting moiety is added to either the 5′ or 3′ end of the sense strand of the iRNA agent.

In a preferred embodiment an asymmetrically modified iRNA agent has a modification on the sense strand which modification is not found on the antisense strand and the antisense strand has a modification which is not found on the sense strand.

Each strand can include one or more asymmetrical modifications. By way of example: one strand can include a first asymmetrical modification which confers a first property on the iRNA agent and the other strand can have a second asymmetrical modification which confers a second property on the iRNA. E.g., one strand, e.g., the sense strand can have a modification which targets the iRNA agent to a tissue, and the other strand, e.g., the antisense strand, has a modification which promotes hybridization with the target gene sequence.

In some embodiments both strands can be modified to optimize the same property, e.g., to increase resistance to nucleolytic degradation, but different modifications are chosen for the sense and the antisense strands, e.g., because the modifications affect other properties as well. E.g., since some changes can affect RISC activity these modifications are chosen for the sense strand.

In an embodiment one strand has an asymmetrical 2′ modification, e.g., a 2′ OMe modification, and the other strand has an asymmetrical modification of the phosphate backbone, e.g., a phosphorothioate modification. So, in one embodiment the antisense strand has an asymmetrical 2′ OMe modification and the sense strand has an asymmetrical phosphorothioate modification (or vice versa). In a particularly preferred embodiment the RNAi agent will have asymmetrical 2′-O alkyl, preferably, 2′-OMe modifications on the sense strand and asymmetrical backbone P modification, preferably a phosphothioate modification in the antisense strand. There can be one or multiple 2′-OMe modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the sense strand can be so modified. There can be one or multiple phosphorothioate modifications, e.g., at least 2, 3, 4, 5, or 6, of the subunits of the antisense strand can be so modified. It is preferable to have an iRNA agent wherein there are multiple 2′-OMe modifications on the sense strand and multiple phophorothioate modifications on the antisense strand. All of the subunits on one or both strands can be so modified. A particularly preferred embodiment of multiple asymmetric modification on both strands has a duplex region about 20-21, and preferably 19, subunits in length and one or two 3′ overhangs of about 2 subunits in length.

Asymmetrical modifications are useful for promoting resistance to degradation by nucleases, e.g., endonucleases. iRNA agents can include one or more asymmetrical modifications which promote resistance to degradation. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Most if not all sites on a strand are vulnerable, to some degree, to degradation by endonucleases. One can determine sites which are relatively vulnerable and insert asymmetrical modifications which inhibit degradation. It is often desirable to provide asymmetrical modification of a UA site in an iRNA agent, and in some cases it is desirable to provide the UA sequence on both strands with asymmetrical modification. Examples of modifications which inhibit endonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety on the U, especially on a sense strand; modification of the backbone, e.g., with the replacement of an O with an S, in the phosphate backbone, e.g., the provision of a phosphorothioate modification, on the U or the A or both, especially on an antisense strand; replacement of the U with a C5 amino linker; replacement of the A with a G (sequence changes are preferred to be located on the sense strand and not the antisense strand); and modification of the at the 2′, 6′, 7′, or 8′ position. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

Asymmetrical modification can be used to inhibit degradation by exonucleases. Asymmetrical modifications can include those in which only one strand is modified as well as those in which both are modified. In preferred embodiments the modification on the antisense strand is one which will not interfere with silencing of the target, e.g., one which will not interfere with cleavage of the target. Some embodiments will have an asymmetrical modification on the sense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus, and on the antisense strand, e.g., in a 3′ overhang, e.g., at the 3′ terminus. If the modifications introduce moieties of different size it is preferable that the larger be on the sense strand. If the modifications introduce moieties of different charge it is preferable that the one with greater charge be on the sense strand.

Examples of modifications which inhibit exonucleolytic degradation can be found herein. Particularly favored modifications include: 2′ modification, e.g., provision of a 2′ OMe moiety in a 3′ overhang, e.g., at the 3′ terminus (3′ terminus means at the 3′ atom of the molecule or at the most 3′ moiety, e.g., the most 3′ P or 2′ position, as indicated by the context); modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P in a 3′ overhang, e.g., at the 3′ terminus; combination of a 2′ modification, e.g., provision of a 2′ O Me moiety and modification of the backbone, e.g., with the replacement of a P with an S, e.g., the provision of a phosphorothioate modification, or the use of a methylated P, in a 3′ overhang, e.g., at the 3′ terminus; modification with a 3′ alkyl; modification with an abasic pyrolidine in a 3′ overhang, e.g., at the 3′ terminus; modification with naproxene, ibuprofen, or other moieties which inhibit degradation at the 3′ terminus. Preferred embodiments are those in which one or more of these modifications are present on the sense but not the antisense strand, or embodiments where the antisense strand has fewer of such modifications.

Modifications, e.g., those described herein, which affect targeting can be provided as asymmetrical modifications. Targeting modifications which can inhibit silencing, e.g., by inhibiting cleavage of a target, can be provided as asymmetrical modifications of the sense strand. A biodistribution altering moiety, e.g., cholesterol, can be provided in one or more, e.g., two, asymmetrical modifications of the sense strand. Targeting modifications which introduce moieties having a relatively large molecular weight, e.g., a molecular weight of more than 400, 500, or 1000 daltons, or which introduce a charged moiety (e.g., having more than one positive charge or one negative charge) can be placed on the sense strand.

Modifications, e.g., those described herein, which modulate, e.g., increase or decrease, the affinity of a strand for its compliment or target, can be provided as asymmetrical modifications. These include: 5 methyl U; 5 methyl C; pseudouridine, Locked nucleic acids ,2 thio U and 2-amino-A. In some embodiments one or more of these is provided on the antisense strand.

iRNA agents have a defined structure, with a sense strand and an antisense strand, and in many cases short single strand overhangs, e.g., of 2 or 3 nucleotides are present at one or both 3′ ends. Asymmetrical modification can be used to optimize the activity of such a structure, e.g., by being placed selectively within the iRNA. E.g., the end region of the iRNA agent defined by the 5′ end of the sense strand and the 3′ end of the antisense strand is important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. In preferred embodiments asymmetrical modifications which result in one or more of the following are used: modifications of the 5′ end of the sense strand which inhibit kinase activation of the sense strand, including, e.g., attachments of conjugates which target the molecule or the use modifications which protect against 5′ exonucleolytic degradation; or modifications of either strand, but preferably the sense strand, which enhance binding between the sense and antisense strand and thereby promote a “tight” structure at this end of the molecule.

The end region of the iRNA agent defined by the 3′ end of the sense strand and the 5′ end of the antisense strand is also important for function. This region can include the terminal 2, 3, or 4 paired nucleotides and any 3′ overhang. Preferred embodiments include asymmetrical modifications of either strand, but preferably the sense strand, which decrease binding between the sense and antisense strand and thereby promote an “open” structure at this end of the molecule. Such modifications include placing conjugates which target the molecule or modifications which promote nuclease resistance on the sense strand in this region. Modification of the antisense strand which inhibit kinase activation are avoided in preferred embodiments.

Exemplary modifications for asymmetrical placement in the sense strand include the following:

(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S); these modifications can be used to promote nuclease resistance;

(b) 2′-O alkyl, e.g., 2′-OMe, 3′-O alkyl, e.g., 3′-OMe (at terminal and/or internal positions); these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand, the 3′ modifications can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe and with P═O or P═S) these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(d) L sugars (e.g., L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(e) modified sugars (e.g., locked nucleic acids (LNA's), hexose nucleic acids (HNA's) and cyclohexene nucleic acids (CeNA's)); these modifications can be used to promote nuclease resistance or to inhibit binding of the sense to the antisense strand, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines), these modifications can be used to promote nuclease resistance or to enhance binding of the sense to the antisense strand;

(g) cationic groups and Zwitterionic groups (preferably at a terminus), these modifications can be used to promote nuclease resistance;

(h) conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates; these modifications can be used to promote nuclease resistance or to target the molecule, or can be used at the 5′ end of the sense strand to avoid sense strand activation by RISC.

Exemplary modifications for asymmetrical placement in the antisense strand include the following:

(a) backbone modifications, e.g., modification of a backbone P, including replacement of P with S, or P substituted with alkyl or allyl, e.g., Me, and dithioates (S—P═S);

(b) 2′-O alkyl, e.g., 2′-OMe, (at terminal positions);

(c) 2′-5′ linkages (with 2′-H, 2′-OH and 2′-OMe) e.g., terminal at the 3′ end); e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with RISC enzyme activity such as kinase activity;

(d) L sugars (e.g, L ribose, L-arabinose with 2′-H, 2′-OH and 2′-OMe); e.g., terminal at the 3′ end; e.g., with P═O or P═S preferably at the 3′-end, these modifications are preferably excluded from the 5′ end region as they may interfere with kinase activity;

(e) modified sugars (e.g., LNA's, HNA's and CeNA's); these modifications are preferably excluded from the 5′ end region as they may contribute to unwanted enhancements of paring between the sense and antisense strands, it is often preferred to have a “loose” structure in the 5′ region, additionally, they may interfere with kinase activity;

(f) nucleobase modifications (e.g., C-5 modified pyrimidines, N-2 modified purines, N-7 modified purines, N-6 modified purines);

(g) cationic groups and Zwitterionic groups (preferably at a terminus);

conjugate groups (preferably at terminal positions), e,g., naproxen, biotin, cholesterol, ibuprofen, folic acid, peptides, and carbohydrates, but bulky groups or generally groups which inhibit RISC activity should are less preferred.

The 5′-OH of the antisense strand should be kept free to promote activity. In some preferred embodiments modifications that promote nuclease resistance should be included at the 3′ end, particularly in the 3′ overhang.

In another aspect, the invention features a method of optimizing, e.g., stabilizing, an iRNA agent. The method includes selecting a sequence having activity, introducing one or more asymmetric modifications into the sequence, wherein the introduction of the asymmetric modification optimizes a property of the iRNA agent but does not result in a decrease in activity.

The decrease in activity can be less than a preselected level of decrease. In preferred embodiments decrease in activity means a decrease of less than 5, 10, 20, 40, or 50% activity, as compared with an otherwise similar iRNA lacking the introduced modification. Activity can, e.g., be measured in vivo, or in vitro, with a result in either being sufficient to demonstrate the required maintenance of activity.

The optimized property can be any property described herein and in particular the properties discussed in the section on asymmetrical modifications provided herein. The modification can be any asymmetrical modification, e.g., an asymmetric modification described in the section on asymmetrical modifications described herein. Particularly preferred asymmetric modifications are 2′-O alkyl modifications, e.g., 2′-OMe modifications, particularly in the sense sequence, and modifications of a backbone O, particularly phosphorothioate modifications, in the antisense sequence.

In a preferred embodiment a sense sequence is selected and provided with an asymmetrical modification, while in other embodiments an antisense sequence is selected and provided with an asymmetrical modification. In some embodiments both sense and antisense sequences are selected and each provided with one or more asymmetrical modifications.

Multiple asymmetric modifications can be introduced into either or both of the sense and antisense sequence. A sequence can have at least 2, 4, 6, 8, or more modifications and all or substantially all of the monomers of a sequence can be modified.

TABLE 2 Some examples of Asymmetric Modification Strand I Strand II Nuclease Resistance (e.g. 2′-OMe) Biodistribution (e.g., P═S) Biodistribution conjugate Protein Binding Functionality (e.g. Lipophile) (e.g. Naproxen) Tissue Distribution Functionality Cell Targeting Functionality (e.g. Carbohydrates) (e.g. Folate for cancer cells) Tissue Distribution Functionality Fusogenic Functionality (e.g. Liver Cell Targeting (e.g. Polyethylene imines) Carbohydrates) Cancer Cell Targeting (e.g. RGD Fusogenic Functionality peptides and imines) (e.g. peptides) Nuclease Resistance (e.g. 2′-OMe) Increase in binding Affinity (5-Me—C, 5-Me—U, 2-thio-U, 2-amino-A, G-clamp, LNA) Tissue Distribution Functionality RISC activity improving Functionality Helical conformation changing Tissue Distribution Functionality Functionalities (P═S; lipophile, carbohydrates)

This table shows examples having strand I with a selected modification and strand II with a selected modification.

Z-X-Y Architecture

In one aspect, the invention features an iRNA agent which can have a Z-X-Y architecture or structure such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/510,246 (Attorney Docket No. 14174-079P02), filed on Oct. 9, 2003, which is hereby incorporated by reference, and in copending, co-owned U.S. Provisional Application Ser. No. 60/510,318 (Attorney Docket No. 14174-079P03), filed on Oct. 10, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents having a Z-X-Y structure and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates a Z-X-Y architecture.

The invention provides an iRNA agent having a first segment, the Z region, a second segment, the X region, and optionally a third region, the Y region:

Z—X—Y.

It may be desirable to modify subunits in one or both of Zand/or Y on one hand and X on the other hand. In some cases they will have the same modification or the same class of modification but it will more often be the case that the modifications made in Z and/or Y will differ from those made in X.

The Z region typically includes a terminus of an iRNA agent. The length of the Z region can vary, but will typically be from 2-14, more preferably 2-10, subunits in length. It typically is single stranded, i.e., it will not base pair with bases of another strand, though it may in some embodiments self associate, e.g., to form a loop structure. Such structures can be formed by the end of a strand looping back and forming an intrastrand duplex. E.g., 2, 3, 4, 5 or more intra-strand bases pairs can form, having a looped out or connecting region, typically of 2 or more subunits which do not pair. This can occur at one or both ends of a strand. A typical embodiment of a Z region is a single strand overhang, e.g., an over hang of the length described elsewhere herein. The Z region can thus be or include a 3′ or 5′ terminal single strand. It can be sense or antisense strand but if it is antisense it is preferred that it is a 3- overhang. Typical inter-subunit bonds in the Z region include: P═O; P═S; S—P═S; P—NR₂; and P—BR₂. Chiral P═X, where X is S, N, or B) inter-subunit bonds can also be present. (These inter-subunit bonds are discussed in more detail elsewhere herein.) Other preferred Z region subunit modifications (also discussed elsewhere herein) can include: 3′-OR, 3′ SR, 2′-OMe, 3′-OMe, and 2′OH modifications and moieties; alpha configuration bases; and 2′ arabino modifications.

The X region will in most cases be duplexed, in the case of a single strand iRNA agent, with a corresponding region of the single strand, or in the case of a double stranded iRNA agent, with the corresponding region of the other strand. The length of the X region can vary but will typically be between 10-45 and more preferably between 15 and 35 subunits. Particularly preferred region X′s will include 17, 18, 19, 29, 21, 22, 23, 24, or 25 nucleotide pairs, though other suitable lengths are described elsewhere herein and can be used. Typical X region subunits include 2′-OH subunits. In typical embodiments phosphate inter-subunit bonds are preferred while phophorothioate or non-phosphate bonds are absent. Other modifications preferred in the X region include: modifications to improve binding, e.g., nucleobase modifications; cationic nucleobase modifications; and C-5 modified pyrimidines, e.g., allylamines. Some embodiments have 4 or more consecutive 2′OH subunits. While the use of phosphorothioate is sometimes non preferred they can be used if they connect less than 4 consecutive 2′OH subunits.

The Y region will generally conform to the the parameters set out for the Z regions. However, the X and Z regions need not be the same, different types and numbers of modifications can be present, and infact, one will usually be a 3′ overhang and one will usually be a 5′ overhang.

In a preferred embodiment the iRNA agent will have a Y and/or Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe or other alkyl; and an X region that includes at least four consecutive ribonucleoside subunits in which the 2′-OH remains unsubstituted.

The subunit linkages (the linkages between subunits) of an iRNA agent can be modified, e.g., to promote resistance to degradation. Numerous examples of such modifications are disclosed herein, one example of which is the phosphorothioate linkage. These modifications can be provided bewteen the subunits of any of the regions, Y, X, and Z. However, it is preferred that their occureceis minimized and in particular it is preferred that consecutive modified linkages be avoided.

In a preferred embodiment the iRNA agent will have a Y and Z region each having ribonucleosides in which the 2′-OH is substituted, e.g., with 2′-OMe; and an X region that includes at least four consecutive subunits, e.g., ribonucleoside subunits in which the 2′-OH remains unsubstituted.

As mentioned above, the subunit linkages of an iRNA agent can be modified, e.g., to promote resistance to degradation. These modifications can be provided between the subunits of any of the regions, Y, X, and Z. However, it is preferred that they are minimized and in particular it is preferred that consecutive modified linkages be avoided.

Thus, in a preferred embodiment, not all of the subunit linkages of the iRNA agent are modified and more preferably the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred iRNA agents will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

It is particularly preferred to minimize the occurrence in region X. Thus, in preferred embodiments each of the nucleoside subunit linkages in X will be phosphodiester linkages, or if subunit linkages in region X are modified, such modifications will be minimized. E.g., although the Y and/or Z regions can include inter subunit linkages which have been stabilized against degradation, such modifications will be minimized in the X region, and in particular consecutive modifications will be minimized. Thus, in preferred embodiments the maximum number of consecutive subunits linked by other than a phospodiester bond will be 2, 3, or 4. Particulary preferred X regions will not have four or more consecutive subunits, e.g., 2′-hydroxyl ribonucleoside subunits, in which each subunits is joined by modified linkages—i.e. linkages that have been modified to stabilize them from degradation as compared to the phosphodiester linkages that naturally occur in RNA and DNA.

In a preferred embodiment Y and /or Z will be free of phosphorothioate linkages, though either or both may contain other modifications, e.g., other modifications of the subunit linkages.

In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical 2′ moieties.

In a preferred embodiment region X, or in some cases, the entire iRNA agent, has no more than 3 or no more than 4 subunits having identical subunit linkages.

In a preferred embodiment one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosphorothioate linkage.

In a preferred embodiment each of Y and/or Z independently has only one phosphorothioate linkage between adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides. If there is a second set of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, in Y and/or Z that second set is connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

In a prefered embodiment each of Y and/orZ independently has more than one phosphorothioate linkage connecting adjacent pairs of subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, but at least one pair of adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., 2′-O-alkyl nucleosides, are be connected by a linkage other than a phosphorothioate linkage, e.g., a modified linkage other than a phosphorothioate linkage.

In a prefered embodiment one of the above recited limitation on adjacent subunits in Y and or Z is combined with a limitation on the subunits in X. E.g., one or more phosphorothioate linkages (or other modifications of the subunit linkage) are present in Y and/or Z, but such modified linkages do not connect two adjacent subunits, e.g., nucleosides, having a 2′ modification, e.g., a 2′-O-alkyl moiety. E.g., any adjacent 2′-O-alkyl moieties in the Y and/or Z, are connected by a linkage other than a a phosporothioate linkage. In addition, the X region has no more than 3 or no more than 4 identical subunits, e.g., subunits having identical 2′ moieties or the X region has no more than 3 or no more than 4 subunits having identical subunit linkages.

A Y and/or Z region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification described herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can be present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively. In a preferred embodiment the frequency of the modification will differ between Yand /or Z and X, e.g., the modification will be present one of Y and/or Z or X and absent in the other.

An X region can include at least one, and preferably 2, 3 or 4 of a modification disclosed herein. Such modifications can be chosen, independently, from any modification desribed herein, e.g., from nuclease resistant subunits, subunits with modified bases, subunits with modified intersubunit linkages, subunits with modified sugars, and subunits linked to another moiety, e.g., a targeting moiety. In a preferred embodiment more than 1 of such subunits can b present but in some emobodiments it is prefered that no more than 1, 2, 3, or 4 of such modifications occur, or occur consecutively.

An RRMS (described elswhere herein) can be introduced at one or more points in one or both strands of a double-stranded iRNA agent. An RRMS can be placed in a Y and/or Z region, at or near (within 1, 2, or 3 positions) of the 3′ or 5′ end of the sense strand or at near (within 2 or 3 positions of) the 3′ end of the antisense strand. In some embodiments it is preferred to not have an RRMS at or near (within 1, 2, or 3 positions of) the 5′ end of the antisense strand. An RRMS can be positioned in the X region, and will preferably be positioned in the sense strand or in an area of the antisense strand not critical for antisense binding to the target.

Differential Modification of Terminal Duplex Stability

In one aspect, the invention features an iRNA agent which can have differential modification of terminal duplex stability (DMTDS).

In addition, the invention includes iRNA agents having DMTDS and another element described herein. E.g., the invention includes an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent having a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having an architecture or structure described herein, an iRNA associated with an amphipathic delivery agent described herein, an iRNA associated with a drug delivery module described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, which also incorporates DMTDS.

iRNA agents can be optimized by increasing the propensity of the duplex to disassociate or melt (decreasing the free energy of duplex association), in the region of the 5′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which increase the propensity of the duplex to disassociate or melt in the region of the 5′ end of the antisense strand. It can also be accomplished by the attachment of a ligand that increases the propensity of the duplex to disassociate of melt in the region of the 5′ end . While not wishing to be bound by theory, the effect may be due to promoting the effect of an enzyme such as helicase, for example, promoting the effect of the enzyme in the proximity of the 5′ end of the antisense strand.

The inventors have also discovered that iRNA agents can be optimized by decreasing the propensity of the duplex to disassociate or melt (increasing the free energy of duplex association), in the region of the 3′ end of the antisense strand duplex. This can be accomplished, e.g., by the inclusion of subunits which decrease the propensity of the duplex to disassociate or melt in the region of the 3′ end of the antisense strand. It can also be accomplished by the attachment of ligand that decreases the propensity of the duplex to disassociate of melt in the region of the 5′ end.

Modifications which increase the tendency of the 5′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which decrease the tendency of the 3′ end of the duplex to dissociate.

Likewise, modifications which decrease the tendency of the 3′ end of the duplex to dissociate can be used alone or in combination with other modifications described herein, e.g., with modifications which increase the tendency of the 5′ end of the duplex to dissociate.

Decreasing the stability of the AS 5′ end of the duplex

Subunit pairs can be ranked on the basis of their propensity to promote dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting dissociation:

A:U is preferred over G:C;

G:U is preferred over G:C;

I:C is preferred over G:C (I=inosine);

mismatches, e.g., non-canonical or other than canonical pairings (as described elsewhere herein) are preferred over canonical (A:T, A:U, G:C) pairings;

pairings which include a universal base are preferred over canonical pairings.

A typical ds iRNA agent can be diagrammed as follows:

S 5′ R₁ N₁ N₂ N₃ N₄ N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₂ 3′

AS 3′ R₃ N₁ N₂ N₃ N₄ N₅ [N] N⁻⁵ N⁻⁴ N⁻³ N⁻² N⁻¹ R₄ 5′

S:AS P₁ P₂ P₃ P₄ P₅ [N] P⁻⁵ P⁻⁴ P⁻³ P⁻² P⁻¹ 5′

S indicates the sense strand; AS indicates antisense strand; R₁ indicates an optional (and nonpreferred) 5′ sense strand overhang; R₂ indicates an optional (though preferred) 3′ sense overhang; R₃ indicates an optional (though preferred) 3′ antisense sense overhang; R₄ indicates an optional (and nonpreferred) 5′ antisense overhang; N indicates subunits; [N] indicates that additional subunit pairs may be present; and P,, indicates a paring of sense N_(x) and antisense N_(x). Overhangs are not shown in the P diagram. In some embodiments a 3′ AS overhang corresponds to region Z, the duplex region corresponds to region X, and the 3′ S strand overhang corresponds to region Y, as described elsewhere herein. (The diagram is not meant to imply maximum or minimum lengths, on which guidance is provided elsewhere herein.)

It is preferred that pairings which decrease the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 5′ end of the AS strand. The terminal pair (the most 5′ pair in terms of the AS strand) is designated as P⁻¹, and the subsequent pairing positions (going in the 3′ direction in terms of the AS strand) in the duplex are designated, P⁻², P⁻³, P⁻⁴, P⁻⁵, and so on. The preferred region in which to modify to modulate duplex formation is at P⁻⁵ through P⁻¹, more preferably P⁻⁴ through P⁻¹, more preferably P⁻³ through P⁻¹. Modification at P⁻¹, is particularly preferred, alone or with modification(s) other position(s), e.g., any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions be chosen independently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairings or pairings which include a universal base.

In preferred embodiments the change in subunit needed to achieve a pairing which promotes dissociation will be made in the sense strand, though in some embodiments the change will be made in the antisense strand.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are pairs which promote disociation.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are G:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are I:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are mismatched pairs, e.g., non-canonical or other than canonical pairings pairings.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through R⁻⁴, are pairings which include a universal base.

Increasing the Stability of the AS 3′ End of the Duplex

Subunit pairs can be ranked on the basis of their propensity to promote stability and inhibit dissociation or melting (e.g., on the free energy of association or dissociation of a particular pairing, the simplest approach is to examine the pairs on an individual pair basis, though next neighbor or similar analysis can also be used). In terms of promoting duplex stability:

G:C is preferred over A:U

Watson-Crick matches (A:T, A:U, G:C) are preferred over non-canonical or other than canonical pairings

analogs that increase stability are preferred over Watson-Crick matches (A:T, A:U, G:C)

2-amino-A:U is preferred over A:U

2-thio U or 5 Me-thio-U:A are preferred over U:A

G-clamp (an analog of C having 4 hydrogen bonds):G is preferred over C:G

guanadinium-G-clamp:G is preferred over C:G

psuedo uridine:A is preferred over U:A

sugar modifications, e.g., 2′ modifications, e.g., 2′F, ENA, or LNA, which enhance binding are preferred over non-modified moieties and can be present on one or both strands to enhance stability of the duplex. It is preferred that pairings which increase the propensity to form a duplex are used at 1 or more of the positions in the duplex at the 3′ end of the AS strand. The terminal pair (the most 3′ pair in terms of the AS strand) is designated as P₁, and the subsequent pairing positions (going in the 5′ direction in terms of the AS strand) in the duplex are designated, P₂, P₃, P₄, P₅, and so on. The preferred region in which to modify to modulate duplex formation is at P₅ through P₁, more preferably P₄ through P₁ , more preferably P₃ through P₁. Modification at P₁, is particularly preferred, alone or with mdification(s) at other position(s), e.g.,any of the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of the recited regions be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

In a preferred embodiment the at least 2, or 3, of the pairs in P⁻¹, through P⁻⁴, are pairs which promote duplex stability.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are G:C.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are a pair having an analog that increases stability over Watson-Crick matches.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are 2-amino-A:U.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are 2-thio U or 5 Me-thio-U:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are guanidinium-G-clamp:G.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are psuedo uridine:A.

In a preferred embodiment the at least 2, or 3, of the pairs in P₁, through P₄, are a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhances binding.

G-clamps and guanidinium G-clamps are discussed in the following references: Holmes and Gait, “The Synthesis of 2′-O-Methyl G-Clamp Containing Oligonucleotides and Their Inhibition of the HIV-1 Tat-TAR Interaction,” Nucleosides, Nucleotides & Nucleic Acids, 22:1259-1262, 2003; Holmes et al., “Steric inhibition of human immunodeficiency virus type-1 Tat-dependent trans-activation in vitro and in cells by oligonucleotides containing 2′-O-methyl G-clamp ribonucleoside analogues,” Nucleic Acids Research, 31:2759-2768, 2003; Wilds, et al., “Structural basis for recognition of guanosine by a synthetic tricyclic cytosine analogue: Guanidinium G-clamp,” Helvetica Chimica Acta, 86:966-978, 2003; Raj eev, et al., “High-Affinity Peptide Nucleic Acid Oligomers Containing Tricyclic Cytosine Analogues,” Organic Letters, 4:4395-4398, 2002; Ausin, et al., “Synthesis of Amino- and Guanidino-G-Clamp PNA Monomers,” Organic Letters, 4:4073-4075, 2002; Maier et al., “Nuclease resistance of oligonucleotides containing the tricyclic cytosine analogues phenoxazine and 9-(2-aminoethoxy)-phenoxazine (”G-clamp“) and origins of their nuclease resistance properties,” Biochemistry, 41:1323-7, 2002; Flanagan, et al., “A cytosine analog that confers enhanced potency to antisense oligonucleotides,” Proceedings Of The National Academy Of Sciences Of The United States Of America, 96:3513-8, 1999.

Simultaneously Decreasing the Stability of the AS 5′ end of the Duplex and Increasing the Stability of the AS 3′ End of the Duplex

As is discussed above, an iRNA agent can be modified to both decrease the stability of the AS 5′ end of the duplex and increase the stability of the AS 3′ end of the duplex. This can be effected by combining one or more of the stability decreasing modifications in the AS 5′ end of the duplex with one or more of the stability increasing modifications in the AS 3′ end of the duplex. Accordingly a preferred embodiment includes modification in P⁻⁵ through P⁻¹, more preferably P⁻⁴ through P⁻¹ and more preferably P⁻³ through P⁻¹. Modification at P⁻¹, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 5′ end of the duplex region be chosen independently from the group of:

A:U

G:U

I:C

mismatched pairs, e.g., non-canonical or other than canonical pairings which include a universal base; and

a modification in P₅ through P₁, more preferably P₄ through P₁ and more preferably P₃ through P₁. Modification at P₁, is particularly preferred, alone or with other position, e.g., the positions just identified. It is preferred that at least 1, and more preferably 2, 3, 4, or 5 of the pairs of one of the recited regions of the AS 3′ end of the duplex region be chosen independently from the group of:

G:C

a pair having an analog that increases stability over Watson-Crick matches (A:T, A:U, G:C)

2-amino-A:U

2-thio U or 5 Me-thio-U:A

G-clamp (an analog of C having 4 hydrogen bonds):G

guanadinium-G-clamp:G

psuedo uridine:A

a pair in which one or both subunits has a sugar modification, e.g., a 2′ modification, e.g., 2′F, ENA, or LNA, which enhance binding.

The invention also includes methods of selecting and making iRNA agents having DMTDS. E.g., when screening a target sequence for candidate sequences for use as iRNA agents one can select sequences having a DMTDS property described herein or one which can be modified, preferably with as few changes as possible, especially to the

AS strand, to provide a desired level of DMTDS.

The invention also includes, providing a candidate iRNA agent sequence, and modifying at least one P in P⁻⁵ through P⁻¹ and/or at least one P in P₅ through P₁ to provide a DMTDS iRNA agent.

DMTDS iRNA agents can be used in any method described herein, e.g., to silence any gene disclosed herein, to treat any disorder described herein, in any formulation described herein, and generally in and/or with the methods and compositions described elsewhere herein. DMTDS iRNA agents can incorporate other modifications described herein, e.g., the attachment of targeting agents or the inclusion of modifications which enhance stability, e.g., the inclusion of nuclease resistant monomers or the inclusion of single strand overhangs (e.g., 3′ AS overhangs and/or 3′ S strand overhangs) which self associate to form intrastrand duplex structure.

Preferably these iRNA agents will have an architecture described herein.

Other Embodiments

In Vivo Delivery

An iRNA agent can be linked, e.g., noncovalently linked to a polymer for the efficient delivery of the iRNA agent to a subject, e.g., a mammal, such as a human. The iRNA agent can, for example, be complexed with cyclodextrin. Cyclodextrins have been used as delivery vehicles of therapeutic compounds. Cyclodextrins can form inclusion complexes with drugs that are able to fit into the hydrophobic cavity of the cyclodextrin. In other examples, cyclodextrins form non-covalent associations with other biologically active molecules such as oligonucleotides and derivatives thereof. The use of cyclodextrins creates a water-soluble drug delivery complex, that can be modified with targeting or other functional groups. Cyclodextrin cellular delivery system for oligonucleotides described in U.S. Pat. No. 5,691,316, which is hereby incorporated by reference, are suitable for use in methods of the invention. In this system, an oligonucleotide is noncovalently complexed with a cyclodextrin, or the oligonucleotide is covalently bound to adamantine which in turn is non-covalently associated with a cyclodextrin.

The delivery molecule can include a linear cyclodextrin copolymer or a linear oxidized cyclodextrin copolymer having at least one ligand bound to the cyclodextrin copolymer. Delivery systems , as described in U.S. Pat. No. 6,509,323, herein incorporated by reference, are suitable for use in methods of the invention. An iRNA agent can be bound to the linear cyclodextrin copolymer and/or a linear oxidized cyclodextrin copolymer. Either or both of the cyclodextrin or oxidized cyclodextrin copolymers can be crosslinked to another polymer and/or bound to a ligand.

A composition for iRNA delivery can employ an “inclusion complex,” a molecular compound having the characteristic structure of an adduct. In this structure, the “host molecule” spatially encloses at least part of another compound in the delivery vehicle. The enclosed compound (the “guest molecule”) is situated in the cavity of the host molecule without affecting the framework structure of the host. A “host” is preferably cyclodextrin, but can be any of the molecules suggested in U.S. Patent Publ. 2003/0008818, herein incorporated by reference.

Cyclodextrins can interact with a variety of ionic and molecular species, and the resulting inclusion compounds belong to the class of “host-guest” complexes. Within the host-guest relationship, the binding sites of the host and guest molecules should be complementary in the stereoelectronic sense. A composition of the invention can contain at least one polymer and at least one therapeutic agent, generally in the form of a particulate composite of the polymer and therapeutic agent, e.g., the iRNA agent. The iRNA agent can contain one or more complexing agents. At least one polymer of the particulate composite can interact with the complexing agent in a host-guest or a guest-host interaction to form an inclusion complex between the polymer and the complexing agent. The polymer and, more particularly, the complexing agent can be used to introduce functionality into the composition. For example, at least one polymer of the particulate composite has host functionality and forms an inclusion complex with a complexing agent having guest functionality. Alternatively, at least one polymer of the particulate composite has guest functionality and forms an inclusion complex with a complexing agent having host functionality. A polymer of the particulate composite can also contain both host and guest functionalities and form inclusion complexes with guest complexing agents and host complexing agents. A polymer with functionality can, for example, facilitate cell targeting and/or cell contact (e.g., targeting or contact to a liver cell), intercellular trafficking, and/or cell entry and release.

Upon forming the particulate composite, the iRNA agent may or may not retain its biological or therapeutic activity. Upon release from the therapeutic composition, specifically, from the polymer of the particulate composite, the activity of the iRNA agent is restored. Accordingly, the particulate composite advantageously affords the iRNA agent protection against loss of activity due to, for example, degradation and offers enhanced bioavailability. Thus, a composition may be used to provide stability, particularly storage or solution stability, to an iRNA agent or any active chemical compound. The iRNA agent may be further modified with a ligand prior to or after particulate composite or therapeutic composition formation. The ligand can provide further functionality. For example, the ligand can be a targeting moiety.

Physiological Effects

The iRNA agents described herein can be designed such that determining therapeutic toxicity is made easier by the complementarity of the iRNA agent with both a human and a non-human animal sequence. By these methods, an iRNA agent can consist of a sequence that is fully complementary to a nucleic acid sequence from a human and a nucleic acid sequence from at least one non-human animal, e.g., a non-human mammal, such as a rodent, ruminant or primate. For example, the non-human mammal can be a mouse, rat, dog, pig, goat, sheep, cow, monkey, Pan paniscus, Pan troglodytes, Macaca mulatto, or Cynomolgus monkey. The sequence of the iRNA agent could be complementary to sequences within homologous genes, e.g., oncogenes or tumor suppressor genes, of the non-human mammal and the human. By determining the toxicity of the iRNA agent in the non-human mammal, one can extrapolate the toxicity of the iRNA agent in a human. For a more strenuous toxicity test, the iRNA agent can be complementary to a human and more than one, e.g., two or three or more, non-human animals.

The methods described herein can be used to correlate any physiological effect of an iRNA agent on a human, e.g., any unwanted effect, such as a toxic effect, or any positive, or desired effect.

Delivery Module

In one aspect, the invention features a drug delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/454,265, filed on Mar. 12, 2003, which is hereby incorporated by reference.

In addition, the invention includes iRNA agents described herein, e.g., a palindromic iRNA agent, an iRNA agent hying a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such a drug delivery conjugate or module.

The iRNA agents can be complexed to a delivery agent that features a modular complex. The complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of): (a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic or electrostatic interactions); (b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane); and (c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell or bone cell.

An iRNA agent, e.g., iRNA agent or sRNA agent described herein, can be linked, e.g., coupled or bound, to the modular complex. The iRNA agent can interact with the condensing agent of the complex, and the complex can be used to deliver an iRNA agent to a cell, e.g., in vitro or in vivo. For example, the complex can be used to deliver an iRNA agent to a subject in need thereof, e.g., to deliver an iRNA agent to a subject having a disorder, e.g., a disorder described herein, such as a disease or disorder of the liver.

The fusogenic agent and the condensing agent can be different agents or the one and the same agent. For example, a polyamino chain, e.g., polyethyleneimine (PEI), can be the fusogenic and/or the condensing agent.

The delivery agent can be a modular complex. For example, the complex can include a carrier agent linked to one or more of (preferably two or more, more preferably all three of):

(a) a condensing agent (e.g., an agent capable of attracting, e.g., binding, a nucleic acid, e.g., through ionic interaction),

(b) a fusogenic agent (e.g., an agent capable of fusing and/or being transported through a cell membrane, e.g., an endosome membrane), and

(c) a targeting group, e.g., a cell or tissue targeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g., an antibody, that binds to a specified cell type such as a cancer cell, endothelial cell, bone cell. A targeting group can be a thyrotropin, melanotropin, lectin, glycoprotein, surfactant protein A, Mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-gulucosamine multivalent mannose, multivalent fucose, glycosylated polyaminoacids, multivalent galactose, transferrin, bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folate, vitamin B12, biotin, Neproxin, or an RGD peptide or RGD peptide mimetic.

Carrier Agents

The carrier agent of a modular complex described herein can be a substrate for attachment of one or more of: a condensing agent, a fusogenic agent, and a targeting group. The carrier agent would preferably lack an endogenous enzymatic activity. The agent would preferably be a biological molecule, preferably a macromolecule. Polymeric biological carriers are preferred. It would also be preferred that the carrier molecule be biodegradable..

The carrier agent can be a naturally occurring substance, such as a protein (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan, inulin, cyclodextrin or hyaluronic acid); or lipid. The carrier molecule can also be a recombinant or synthetic molecule, such as a synthetic polymer, e.g., a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic acid anhydride copolymer, poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, or polyphosphazine. Other useful carrier molecules can be identified by routine methods.

A carrier agent can be characterized by one or more of: (a) is at least 1 Da in size; (b) has at least 5 charged groups, preferably between 5 and 5000 charged groups; (c) is present in the complex at a ratio of at least 1:1 carrier agent to fusogenic agent; (d) is present in the complex at a ratio of at least 1:1 carrier agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 carrier agent to targeting agent.

Fusogenic Agents

A fusogenic agent of a modular complex described herein can be an agent that is responsive to, e.g., changes charge depending on, the pH environment. Upon encountering the pH of an endosome, it can cause a physical change, e.g., a change in osmotic properties which disrupts or increases the permeability of the endosome membrane. Preferably, the fusogenic agent changes charge, e.g., becomes protonated, at pH lower than physiological range. For example, the fusogenic agent can become protonated at pH 4.5-6.5. The fusogenic agent can serve to release the iRNA agent into the cytoplasm of a cell after the complex is taken up, e.g., via endocytosis, by the cell, thereby increasing the cellular concentration of the iRNA agent in the cell.

In one embodiment, the fusogenic agent can have a moiety, e.g., an amino group, which, when exposed to a specified pH range, will undergo a change, e.g., in charge, e.g., protonation. The change in charge of the fusogenic agent can trigger a change, e.g., an osmotic change, in a vesicle, e.g., an endocytic vesicle, e.g., an endosome. For example, the fusogenic agent, upon being exposed to the pH environment of an endosome, will cause a solubility or osmotic change substantial enough to increase the porosity of (preferably, to rupture) the endosomal membrane.

The fusogenic agent can be a polymer, preferably a polyamino chain, e.g., polyethyleneimine (PEI). The PEI can be linear, branched, synthetic or natural. The PEI can be, e.g., alkyl substituted PEI, or lipid substituted PEI.

In other embodiments, the fusogenic agent can be polyhistidine, polyimidazole, polypyridine, polypropyleneimine, mellitin, or a polyacetal substance, e.g., a cationic polyacetal. In some embodiment, the fusogenic agent can have an alpha helical structure. The fusogenic agent can be a membrane disruptive agent, e.g., mellittin. A fusogenic agent can have one or more of the following characteristics: (a) is at least 1 Da in size; (b) has at least 10 charged groups, preferably between 10 and 5000 charged groups, more preferably between 50 and 1000 charged groups; (c) is present in the complex at a ratio of at least 1:1 fusogenic agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 fusogenic agent to condensing agent; (e) is present in the complex at a ratio of at least 1:1 fusogenic agent to targeting agent.

Other suitable fusogenic agents can be tested and identified by a skilled artisan. The ability of a compound to respond to, e.g., change charge depending on, the pH environment can be tested by routine methods, e.g., in a cellular assay. For example, a test compound is combined or contacted with a cell, and the cell is allowed to take up the test compound, e.g., by endocytosis. An endosome preparation can then be made from the contacted cells and the endosome preparation compared to an endosome preparation from control cells. A change, e.g., a decrease, in the endosome fraction from the contacted cell vs. the control cell indicates that the test compound can function as a fusogenic agent. Alternatively, the contacted cell and control cell can be evaluated, e.g., by microscopy, e.g., by light or electron microscopy, to determine a difference in endosome population in the cells. The test compound can be labeled. In another type of assay, a modular complex described herein is constructed using one or more test or putative fusogenic agents. The modular complex can be constructed using a labeled nucleic acid instead of the iRNA. The ability of the fusogenic agent to respond to, e.g., change charge depending on, the pH environment, once the modular complex is taken up by the cell, can be evaluated, e.g., by preparation of an endosome preparation, or by microscopy techniques, as described above. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to, e.g., change charge depending on, the pH environment; and a second assay evaluates the ability of a modular complex that includes the test compound to respond to, e.g., change charge depending on, the pH environment.

Condensing Agent

The condensing agent of a modular complex described herein can interact with (e.g., attracts, holds, or binds to) an iRNA agent and act to (a) condense, e.g., reduce the size or charge of the iRNA agent and/or (b) protect the iRNA agent, e.g., protect the iRNA agent against degradation. The condensing agent can include a moiety, e.g., a charged moiety, that can interact with a nucleic acid, e.g., an iRNA agent, e.g., by ionic interactions. The condensing agent would preferably be a charged polymer, e.g., a polycationic chain. The condensing agent can be a polylysine (PLL), spermine, spermidine, polyamine, pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine, arginine, amidine, protamine, cationic lipid, cationic porphyrin, quarternary salt of a polyamine, or an alpha helical peptide.

A condensing agent can have the following characteristics: (a) at least 1Da in size; (b) has at least 2 charged groups, preferably between 2 and 100 charged groups; (c) is present in the complex at a ratio of at least 1:1 condensing agent to carrier agent; (d) is present in the complex at a ratio of at least 1:1 condensing agent to fusogenic agent; (e) is present in the complex at a ratio of at least 1:1 condensing agent to targeting agent.

Other suitable condensing agents can be tested and identified by a skilled artisan, e.g., by evaluating the ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent. The ability of a test agent to interact with a nucleic acid, e.g., an iRNA agent, e.g., to condense or protect the iRNA agent, can be evaluated by routine techniques. In one assay, a test agent is contacted with a nucleic acid, and the size and/or charge of the contacted nucleic acid is evaluated by a technique suitable to detect changes in molecular mass and/or charge. Such techniques include non-denaturing gel electrophoresis, immunological methods, e.g., immunoprecipitation, gel filtration, ionic interaction chromatography, and the like. A test agent is identified as a condensing agent if it changes the mass and/or charge (preferably both) of the contacted nucleic acid, compared to a control. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic cid; and a second assay evaluates the ability of a modular complex that includes the test compound to interact with, e.g., bind to, e.g., condense the charge and/or mass of, a nucleic acid.

Amphipathic Delivery Agents

In one aspect, the invention features an amphipathic delivery conjugate or module, such as those described herein and those described in copending, co-owned U.S. Provisional Application Ser. No. 60/455,050 (Attorney Docket No. 14174-065P01), filed on Mar. 13, 2003, which is hereby incorporated by reference.

In addition, the invention include an iRNA agent described herein, e.g., a palindromic iRNA agent, an iRNA agent hying a non canonical pairing, an iRNA agent which targets a gene described herein, e.g., a gene active in the liver, an iRNA agent having a chemical modification described herein, e.g., a modification which enhances resistance to degradation, an iRNA agent having an architecture or structure described herein, an iRNA agent administered as described herein, or an iRNA agent formulated as described herein, combined with, associated with, and delivered by such an amphipathic delivery conjugate.

An amphipathic molecule is a molecule having a hydrophobic and a hydrophilic region. Such molecules can interact with (e.g., penetrate or disrupt) lipids, e.g., a lipid bylayer of a cell. As such, they can serve as delivery agent for an associated (e.g., bound) iRNA (e.g., an iRNA or sRNA described herein). A preferred amphipathic molecule to be used in the compositions described herein (e.g., the amphipathic iRNA constructs descriebd herein) is a polymer. The polymer may have a secondary structure, e.g., a repeating secondary structure.

One example of an amphipathic polymer is an amphipathic polypeptide, e.g., a polypeptide having a secondary structure such that the polypeptide has a hydrophilic and a hybrophobic face. The design of amphipathic peptide structures (e.g., alpha-helical polypeptides) is routine to one of skill in the art. For example, the following references provide guidance: Grell et al. (2001) Protein design and folding: template trapping of self-assembled helical bundles J Pept Sci 7(3):146-51; Chen et al. (2002) Determination of stereochemistry stability coefficients of amino acid side-chains in an amphipathic alpha-helix J Pept Res 59(1):18-33; Iwata et al. (1994) Design and synthesis of amphipathic 3(10)-helical peptides and their interactions with phospholipid bilayers and ion channel formation J Biol Chem 269(7):4928-33; Cornut et al. (1994) The amphipathic alpha-helix concept. Application to the de novo design of ideally amphipathic Leu, Lys peptides with hemolytic activity higher than that of melittin FEBS Lett 349(1):29-33; Negrete et al. (1998) Deciphering the structural code for proteins: helical propensities in domain classes and statistical multiresidue information in alpha-helices. Protein Sci 7(6):1368-79.

Another example of an amphipathic polymer is a polymer made up of two or more amphipathic subunits, e.g., two or more subunits containing cyclic moieties (e.g., a cyclic moiety having one or more hydrophilic groups and one or more hydrophobic groups). For example, the subunit may contain a steroid, e.g., cholic acid; or a aromatic moiety. Such moieties preferably can exhibit atropisomerism, such that they can form opposing hydrophobic and hydrophilic faces when in a polymer structure.

The ability of a putative amphipathic molecule to interact with a lipid membrane, e.g., a cell membrane, can be tested by routine methods, e.g., in a cell free or cellular assay. For example, a test compound is combined or contacted with a synthetic lipid bilayer, a cellular membrane fraction, or a cell, and the test compound is evaluated for its ability to interact with, penetrate or disrupt the lipid bilayer, cell membrane or cell. The test compound can labeled in order to detect the interaction with the lipid bilayer, cell membrane or cell. In another type of assay, the test compound is linked to a reporter molecule or an iRNA agent (e.g., an iRNA or sRNA described herein) and the ability of the reporter molecule or iRNA agent to penetrate the lipid bilayer, cell membrane or cell is evaluated. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to interact with a lipid bilayer, cell membrane or cell; and a second assay evaluates the ability of a construct (e.g., a construct described herein) that includes the test compound and a reporter or iRNA agent to interact with a lipid bilayer, cell membrane or cell.

An amphipathic polymer useful in the compositions described herein has at least 2, preferably at least 5, more preferably at least 10, 25, 50, 100, 200, 500, 1000, 2000, 50000 or more subunits (e.g., amino acids or cyclic subunits). A single amphipathic polymer can be linked to one or more, e.g., 2, 3, 5, 10 or more iRNA agents (e.g., iRNA or sRNA agents described herein). In some embodiments, an amphipathic polymer can contain both amino acid and cyclic subunits, e.g., aromatic subunits.

The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.

The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):

The invention features a composition that includes an iRNA agent (e.g., an iRNA or sRNA described herein) in association with an amphipathic molecule. Such compositions may be referred to herein as “amphipathic iRNA constructs.” Such compositions and constructs are useful in the delivery or targeting of iRNA agents, e.g., delivery or targeting of iRNA agents to a cell. While not wanting to be bound by theory, such compositions and constructs can increase the porosity of, e.g., can penetrate or disrupt, a lipid (e.g., a lipid bilayer of a cell), e.g., to allow entry of the iRNA agent into a cell.

In one aspect, the invention relates to a composition comprising an iRNA agent (e.g., an iRNA or sRNA agent described herein) linked to an amphipathic molecule. The iRNA agent and the amphipathic molecule may be held in continuous contact with one another by either covalent or noncovalent linkages.

The amphipathic molecule of the composition or construct is preferably other than a phospholipid, e.g., other than a micelle, membrane or membrane fragment.

The amphipathic molecule of the composition or construct is preferably a polymer. The polymer may include two or more amphipathic subunits. One or more hydrophilic groups and one or more hydrophobic groups may be present on the polymer. The polymer may have a repeating secondary structure as well as a first face and a second face. The distribution of the hydrophilic groups and the hydrophobic groups along the repeating secondary structure can be such that one face of the polymer is a hydrophilic face and the other face of the polymer is a hydrophobic face.

The amphipathic molecule can be a polypeptide, e.g., a polypeptide comprising an α-helical conformation as its secondary structure.

In one embodiment, the amphipathic polymer includes one or more subunits containing one or more cyclic moiety (e.g., a cyclic moiety having one or more hydrophilic groups and/or one or more hydrophobic groups). In one embodiment, the polymer is a polymer of cyclic moieties such that the moieties have alternating hydrophobic and hydrophilic groups. For example, the subunit may contain a steroid, e.g., cholic acid. In another example, the subunit may contain an aromatic moiety. The aromatic moiety may be one that can exhibit atropisomerism, e.g., a 2,2′-bis(substituted)-1-1′-binaphthyl or a 2,2′-bis(substituted) biphenyl. A subunit may include an aromatic moiety of Formula (M):

Referring to Formula M, R₁ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; C₁-C₁₀₀ perfluoroalkyl; or OR₅.

R₂ is hydroxy; nitro; sulfate; phosphate; phosphate ester; sulfonic acid; OR₆; or C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

R₃ is hydrogen, or when taken together with R₄ froms a fused phenyl ring.

R₄ is hydrogen, or when taken together with R₃ froms a fused phenyl ring.

R₅ is C₁-C₁₀₀ alkyl optionally substituted with aryl, alkenyl, alkynyl, alkoxy or halo and/or optionally inserted with O, S, alkenyl or alkynyl; or C₁-C₁₀₀ perfluoroalkyl; and R₆ is C₁-C₁₀₀ alkyl optionally substituted with hydroxy, halo, nitro, aryl or alkyl sulfinyl, aryl or alkyl sulfonyl, sulfate, sulfonic acid, phosphate, phosphate ester, substituted or unsubstituted aryl, carboxyl, carboxylate, amino carbonyl, or alkoxycarbonyl, and/or optionally inserted with O, NH, S, S(O), SO₂, alkenyl, or alkynyl.

Increasing Cellular Uptake of dsRNAs

A method of the invention that can include the administration of an iRNA agent and a drug that affects the uptake of the iRNA agent into the cell. The drug can be administered before, after, or at the same time that the iRNA agent is administered. The drug can be covalently linked to the iRNA agent. The drug can be, for example, a lipopolysaccharide, an activator of p38 MAP kinase, or an activator of NF-κB. The drug can have a transient effect on the cell.

The drug can increase the uptake of the iRNA agent into the cell, for example, by disrupting the cell's cytoskeleton, e.g., by disrupting the cell's microtubules, microfilaments, and/or intermediate filaments. The drug can be, for example, taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

The drug can also increase the uptake of the iRNA agent into the cell by activating an inflammatory response, for example. Exemplary drug's that would have such an effect include tumor necrosis factor alpha (TNFalpha), interleukin-1 beta, or gamma interferon.

iRNA Conjugates

An iRNA agent can be coupled, e.g., covalently coupled, to a second agent. For example, an iRNA agent used to treat a particular disorder can be coupled to a second therapeutic agent, e.g., an agent other than the iRNA agent. The second therapeutic agent can be one which is directed to the treatment of the same disorder. For example, in the case of an iRNA used to treat a disorder characterized by unwanted cell proliferation, e.g., cancer, the iRNA agent can be coupled to a second agent which has an anti-cancer effect. For example, it can be coupled to an agent which stimulates the immune system, e.g., a CpG motif, or more generally an agent that activates a toll-like receptor and/or increases the production of gamma interferon.

iRNA Production

An iRNA can be produced, e.g., in bulk, by a variety of methods. Exemplary methods include: organic synthesis and RNA cleavage, e.g., in vitro cleavage.

Organic Synthesis

An iRNA can be made by separately synthesizing each respective strand of a double-stranded RNA molecule. The component strands can then be annealed.

A large bioreactor, e.g., the OligoPilot II from Pharmacia Biotec AB (Uppsala Sweden), can be used to produce a large amount of a particular RNA strand for a given iRNA. The OligoPilotII reactor can efficiently couple a nucleotide using only a 1.5 molar excess of a phosphoramidite nucleotide. To make an RNA strand, ribonucleotides amidites are used. Standard cycles of monomer addition can be used to synthesize the 21 to 23 nucleotide strand for the iRNA. Typically, the two complementary strands are produced separately and then annealed, e.g., after release from the solid support and deprotection.

Organic synthesis can be used to produce a discrete iRNA species. The complementary of the species to a particular target gene can be precisely specified. For example, the species may be complementary to a region that includes a polymorphism, e.g., a single nucleotide polymorphism. Further the location of the polymorphism can be precisely defined. In some embodiments, the polymorphism is located in an internal region, e.g., at least 4, 5, 7, or 9 nucleotides from one or both of the termini.

dsRNA Cleavage

iRNAs can also be made by cleaving a larger ds iRNA. The cleavage can be mediated in vitro or in vivo. For example, to produce iRNAs by cleavage in vitro, the following method can be used:

In vitro transcription. dsRNA is produced by transcribing a nucleic acid (DNA) segment in both directions. For example, the HiScribeTM RNAi transcription kit (New England Biolabs) provides a vector and a method for producing a dsRNA for a nucleic acid segment that is cloned into the vector at a position flanked on either side by a T7 promoter. Separate templates are generated for T7 transcription of the two complementary strands for the dsRNA. The templates are transcribed in vitro by addition of T7 RNA polymerase and dsRNA is produced. Similar methods using PCR and/or other RNA polymerases (e.g., T3 or SP6 polymerase) can also be used. In one embodiment, RNA generated by this method is carefully purified to remove endotoxins that may contaminate preparations of the recombinant enzymes.

In vitro cleavage. dsRNA is cleaved in vitro into iRNAs, for example, using a Dicer or comparable RNAse III-based activity. For example, the dsRNA can be incubated in an in vitro extract from Drosophila or using purified components, e.g. a purified RNAse or RISC complex (RNA-induced silencing complex). See, e.g., Ketting et al. Genes Dev 2001 Oct. 15; 15(20):2654-9. and Hammond Science 2001 Aug. 10; 293(5532):1146-50.

dsRNA cleavage generally produces a plurality of iRNA species, each being a particular 21 to 23 nt fragment of a source dsRNA molecule. For example, iRNAs that include sequences complementary to overlapping regions and adjacent regions of a source dsRNA molecule may be present.

Regardless of the method of synthesis, the iRNA preparation can be prepared in a solution (e.g., an aqueous and/or organic solution) that is appropriate for formulation. For example, the iRNA preparation can be precipitated and redissolved in pure double-distilled water, and lyophilized. The dried iRNA can then be resuspended in a solution appropriate for the intended formulation process.

Synthesis of modified and nucleotide surrogate iRNA agents is discussed below.

Formulation

The iRNA agents described herein can be formulated for administration to a subject

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.

A formulated iRNA composition can assume a variety of states. In some examples, the composition is at least partially crystalline, uniformly crystalline, and/or anhydrous (e.g., less than 80, 50, 30, 20, or 10% water). In another example, the iRNA is in an aqueous phase, e.g., in a solution that includes water.

The aqueous phase or the crystalline compositions can, e.g., be incorporated into a delivery vehicle, e.g., a liposome (particularly for the aqueous phase) or a particle (e.g., a microparticle as can be appropriate for a crystalline composition). Generally, the iRNA composition is formulated in a manner that is compatible with the intended method of administration (see, below).

In particular embodiments, the composition is prepared by at least one of the following methods: spray drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques; or sonication with a lipid, freeze-drying, condensation and other self-assembly. A iRNA preparation can be formulated in combination with another agent, e.g., another therapeutic agent or an agent that stabilizes a iRNA, e.g., a protein that complexes with iRNA to form an iRNP. Still other agents include chelators, e.g., EDTA (e.g., to remove divalent cations such as Mg²⁻), salts, RNAse inhibitors (e.g., a broad specificity RNAse inhibitor such as RNAsin) and so forth.

In one embodiment, the iRNA preparation includes another iRNA agent, e.g., a second iRNA that can mediated RNAi with respect to a second gene, or with respect to the same gene. Still other preparation can include at least 3, 5, ten, twenty, fifty, or a hundred or more different iRNA species. Such iRNAs can mediated RNAi with respect to a similar number of different genes.

In one embodiment, the iRNA preparation includes at least a second therapeutic agent (e.g., an agent other than an RNA or a DNA). For example, a iRNA composition for the treatment of a viral disease, e.g. HIV, might include a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a iRNA composition for the treatment of a cancer might further comprise a chemotherapeutic agent.

Exemplary formulations are discussed below:

Liposomes

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA s agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be formulated for delivery in a membranous molecular assembly, e.g., a liposome or a micelle. As used herein, the term “liposome” refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer, e.g., one bilayer or a plurality of bilayers. Liposomes include unilamellar and multilamellar vesicles that have a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the iRNA composition. The lipophilic material isolates the aqueous interior from an aqueous exterior, which typically does not include the iRNA composition, although in some examples, it may. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomal bilayer fuses with bilayer of the cellular membranes. As the merging of the liposome and cell progresses, the internal aqueous contents that include the iRNA are delivered into the cell where the iRNA can specifically bind to a target RNA and can mediate RNAi. In some cases the liposomes are also specifically targeted, e.g., to direct the iRNA to particular cell types.

A liposome containing a iRNA can be prepared by a variety of methods.

In one example, the lipid component of a liposome is dissolved in a detergent so that micelles are formed with the lipid component. For example, the lipid component can be an amphipathic cationic lipid or lipid conjugate. The detergent can have a high critical micelle concentration and may be nonionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The iRNA preparation is then added to the micelles that include the lipid component. The cationic groups on the lipid interact with the iRNA and condense around the iRNA to form a liposome. After condensation, the detergent is removed, e.g. , by dialysis, to yield a liposomal preparation of iRNA.

If necessary a carrier compound that assists in condensation can be added during the condensation reaction, e.g., by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). pH can also adjusted to favor condensation.

Further description of methods for producing stable polynucleotide delivery vehicles, which incorporate a polynucleotide/cationic lipid complex as structural components of the delivery vehicle, are described in, e.g., WO 96/37194. Liposome formation can also include one or more aspects of exemplary methods described in Felgner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987; U.S. Pat. No. 4,897,355; U.S. Pat. No. 5,171,678; Bangham, et al. M. Mol. Biol. 23:238, 1965; Olson, et al. Biochim. Biophys. Acta 557:9, 1979; Szoka, et al. Proc. Natl. Acad. Sci. 75: 4194, 1978; Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984; Kim, et al. Biochim. Biophys. Acta 728:339, 1983; and Fukunaga, et al. Endocrinol. 115:757, 1984. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw plus extrusion (see, e.g., Mayer, et al. Biochim. Biophys. Acta 858:161, 1986). Microfluidization can be used when consistently small (50 to 200 nm) and relatively uniform aggregates are desired (Mayhew, et al. Biochim. Biophys. Acta 775:169, 1984). These methods are readily adapted to packaging iRNA preparations into liposomes.

Liposomes that are pH-sensitive or negatively-charged, entrap nucleic acid molecules rather than complex with them. Since both the nucleic acid molecules and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some nucleic acid molecules are entrapped within the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver DNA encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene was detected in the target cells (Zhou et al., Journal of Controlled Release, 19, (1992) 269-274).

One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Examples of other methods to introduce liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185; U.S. Pat. No. 5,171,678; WO 94/00569; WO 93/24640; WO 91/16024; Feigner, J. Biol. Chem. 269:2550, 1994; Nabel, Proc. Natl. Acad. Sci. 90:11307, 1993; Nabel, Human Gene Ther. 3:649, 1992; Gershon, Biochem. 32:7143, 1993; and Strauss EMBO J. 11:417, 1992.

In one embodiment, cationic liposomes are used. Cationic liposomes possess the advantage of being able to fuse to the cell membrane. Non-cationic liposomes, although not able to fuse as efficiently with the plasma membrane, are taken up by macrophages in vivo and can be used to deliver iRNAs to macrophages.

Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated iRNAs in their internal compartments from metabolism and degradation (Rosoff, in “Pharmaceutical Dosage Forms,” Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p. 245). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

A positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA) can be used to form small liposomes that interact spontaneously with nucleic acid to form lipid-nucleic acid complexes which are capable of fusing with the negatively charged lipids of the cell membranes of tissue culture cells, resulting in delivery of iRNA (see, e.g., Feigner, P. L. et al., Proc. Natl. Acad. Sci., USA 8:7413-7417, 1987 and U.S. Pat. No. 4,897,355 for a description of DOTMA and its use with DNA).

A DOTMA analogue, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP) can be used in combination with a phospholipid to form DNA-complexing vesicles. Lipofectin™ Bethesda Research Laboratories, Gaithersburg, Md.) is an effective agent for the delivery of highly anionic nucleic acids into living tissue culture cells that comprise positively charged DOTMA liposomes which interact spontaneously with negatively charged polynucleotides to form complexes. When enough positively charged liposomes are used, the net charge on the resulting complexes is also positive. Positively charged complexes prepared in this way spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids into, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane (“DOTAP”) (Boehringer Mannheim, Indianapolis, Ind.) differs from DOTMA in that the oleoyl moieties are linked by ester, rather than ether linkages.

Other reported cationic lipid compounds include those that have been conjugated to a variety of moieties including, for example, carboxyspermine which has been conjugated to one of two types of lipids and includes compounds such as 5-carboxyspermylglycine dioctaoleoylamide (“DOGS”) (Transfectam™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermyl-amide (“DPPES”) (see, e.g., U.S. Pat. No. 5,171,678).

Another cationic lipid conjugate includes derivatization of the lipid with cholesterol (“DC-Chol”) which has been formulated into liposomes in combination with DOPE (See, Gao, X. and Huang, L., Biochim. Biophys. Res. Commun. 179:280, 1991). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., Biochim. Biophys. Acta 1065:8, 1991). For certain cell lines, these liposomes containing conjugated cationic lipids, are said to exhibit lower toxicity and provide more efficient transfection than the DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for the delivery of oligonucleotides are described in WO 98/39359 and WO 96/37194.

Liposomal formulations are particularly suited for topical administration, liposomes present several advantages over other formulations. Such advantages include reduced side effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer iRNA, into the skin. In some implementations, liposomes are used for delivering iRNA to epidermal cells and also to enhance the penetration of iRNA into dermal tissues, e.g., into skin. For example, the liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been documented (see, e.g., Weiner et al., Journal of Drug Targeting, 1992, vol. 2,405-410 and du Plessis et al., Antiviral Research, 18, 1992, 259-265; Mannino, R. J. and Fould-Fogerite, S., Biotechniques 6:682-690, 1988; Itani, T. et al. Gene 56:267-276. 1987; Nicolau, C. et al. Meth. Enz. 149:157-176, 1987; Straubinger, R. M. and Papahadjopoulos, D. Meth. Enz. 101:512-527, 1983; Wang, C. Y. and Huang, L., Proc. Natl. Acad. Sci. USA 84:7851-7855, 1987).

Non-ionic liposomal systems have also been examined to determine their utility in the delivery of drugs to the skin, in particular systems comprising non-ionic surfactant and cholesterol. Non-ionic liposomal formulations comprising Novasome I (glyceryl dilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome II (glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) were used to deliver a drug into the dermis of mouse skin. Such formulations with iRNA are useful for treating a dermatological disorder.

Liposomes that include iRNA can be made highly deformable. Such deformability can enable the liposomes to penetrate through pore that are smaller than the average radius of the liposome. For example, transfersomes are a type of deformable liposomes. Transferosomes can be made by adding surface edge activators, usually surfactants, to a standard liposomal composition. Transfersomes that include iRNA can be delivered, for example, subcutaneously by infection in order to deliver iRNA to keratinocytes in the skin. In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transdermal gradient. In addition, due to the lipid properties, these transferosomes can be self-optimizing (adaptive to the shape of pores, e.g., in the skin), self-repairing, and can frequently reach their targets without fragmenting, and often self-loading. The iRNA agents can include an RRMS tethered to a moiety which improves association with a liposome.

Surfactants

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes (see above). iRNA (or a precursor, e.g., a larger dsRNA which can be processed into a iRNA, or a DNA which encodes a iRNA or precursor) compositions can include a surfactant. In one embodiment, the iRNA is formulated as an emulsion that includes a surfactant. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group provides the most useful means for categorizing the different surfactants used in formulations (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285). If the surfactant molecule is not ionized, it is classified as a nonionic surfactant.

Nonionic surfactants find wide application in pharmaceutical products and are usable over a wide range of pH values. In general their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, in “Pharmaceutical Dosage Forms,” Marcel Dekker, Inc., New York, N.Y., 1988, p. 285).

Micelles and other Membranous Formulations

For ease of exposition the micelles and other formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these micelles and other formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) composition can be provided as a micellar formulation. “Micelles” are defined herein as a particular type of molecular assembly in which amphipathic molecules are arranged in a spherical structure such that all the hydrophobic portions of the molecules are directed inward, leaving the hydrophilic portions in contact with the surrounding aqueous phase. The converse arrangement exists if the environment is hydrophobic.

A mixed micellar formulation suitable for delivery through transdermal membranes may be prepared by mixing an aqueous solution of the iRNA composition, an alkali metal C₈ to C₂₂ alkyl sulphate, and a micelle forming compounds. Exemplary micelle forming compounds include lecithin, hyaluronic acid, pharmaceutically acceptable salts of hyaluronic acid, glycolic acid, lactic acid, chamomile extract, cucumber extract, oleic acid, linoleic acid, linolenic acid, monoolein, monooleates, monolaurates, borage oil, evening of primrose oil, menthol, trihydroxy oxo cholanyl glycine and pharmaceutically acceptable salts thereof, glycerin, polyglycerin, lysine, polylysine, triolein, polyoxyethylene ethers and analogues thereof, polidocanol alkyl ethers and analogues thereof, chenodeoxycholate, deoxycholate, and mixtures thereof. The micelle forming compounds may be added at the same time or after addition of the alkali metal alkyl sulphate. Mixed micelles will form with substantially any kind of mixing of the ingredients but vigorous mixing is preferred in order to provide smaller size micelles.

In one method a first micellar composition is prepared which contains the iRNA composition and at least the alkali metal alkyl sulphate. The first micellar composition is then mixed with at least three micelle forming compounds to form a mixed micellar composition. In another method, the micellar composition is prepared by mixing the iRNA composition, the alkali metal alkyl sulphate and at least one of the micelle forming compounds, followed by addition of the remaining micelle forming compounds, with vigorous mixing.

Phenol and/or m-cresol may be added to the mixed micellar composition to stabilize the formulation and protect against bacterial growth. Alternatively, phenol and/or m-cresol may be added with the micelle forming ingredients. An isotonic agent such as glycerin may also be added after formation of the mixed micellar composition.

For delivery of the micellar formulation as a spray, the formulation can be put into an aerosol dispenser and the dispenser is charged with a propellant. The propellant, which is under pressure, is in liquid form in the dispenser. The ratios of the ingredients are adjusted so that the aqueous and propellant phases become one, i.e. there is one phase. If there are two phases, it is necessary to shake the dispenser prior to dispensing a portion of the contents, e.g. through a metered valve. The dispensed dose of pharmaceutical agent is propelled from the metered valve in a fine spray.

The preferred propellants are hydrogen-containing chlorofluorocarbons, hydrogen-containing fluorocarbons, dimethyl ether and diethyl ether. Even more preferred is HFA 134a (1,1,1,2 tetrafluoroethane).

The specific concentrations of the essential ingredients can be determined by relatively straightforward experimentation. For absorption through the oral cavities, it is often desirable to increase, e.g. at least double or triple, the dosage for through injection or administration through the gastrointestinal tract.

The iRNA agents can include an RRMS tethered to a moiety which improves association with a micelle or other membranous formulation.

Particles

For ease of exposition the particles, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these particles, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In another embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparations may be incorporated into a particle, e.g., a microparticle. Microparticles can be produced by spray-drying, but may also be produced by other methods including lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination of these techniques. See below for further description.

Sustained -Release Formulations. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein can be formulated for controlled, e.g., slow release. Controlled release can be achieved by disposing the iRNA within a structure or substance which impedes its release. E.g., iRNA can be disposed within a porous matrix or in an erodable matrix, either of which allow release of the iRNA over a period of time.

Polymeric particles, e.g., polymeric in microparticles can be used as a sustained-release reservoir of iRNA that is taken up by cells only released from the microparticle through biodegradation. The polymeric particles in this embodiment should therefore be large enough to preclude phagocytosis (e.g., larger than 10 μm and preferably larger than 20 μm). Such particles can be produced by the same methods to make smaller particles, but with less vigorous mixing of the first and second emulsions. That is to say, a lower homogenization speed, vortex mixing speed, or sonication setting can be used to obtain particles having a diameter around 100 μm rather than 10 82 m. The time of mixing also can be altered.

Larger microparticles can be formulated as a suspension, a powder, or an implantable solid, to be delivered by intramuscular, subcutaneous, intradermal, intravenous, or intraperitoneal injection; via inhalation (intranasal or intrapulmonary); orally; or by implantation. These particles are useful for delivery of any iRNA when slow release over a relatively long term is desired. The rate of degradation, and consequently of release, varies with the polymeric formulation.

Microparticles preferably include pores, voids, hollows, defects or other interstitial spaces that allow the fluid suspension medium to freely permeate or perfuse the particulate boundary. For example, the perforated microstructures can be used to form hollow, porous spray dried microspheres.

Polymeric particles containing iRNA (e.g., a sRNA) can be made using a double emulsion technique, for instance. First, the polymer is dissolved in an organic solvent. A preferred polymer is polylactic-co-glycolic acid (PLGA), with a lactic/glycolic acid weight ratio of 65:35, 50:50, or 75:25. Next, a sample of nucleic acid suspended in aqueous solution is added to the polymer solution and the two solutions are mixed to form a first emulsion. The solutions can be mixed by vortexing or shaking, and in a preferred method, the mixture can be sonicated. Most preferable is any method by which the nucleic acid receives the least amount of damage in the form of nicking, shearing, or degradation, while still allowing the formation of an appropriate emulsion. For example, acceptable results can be obtained with a Vibra-cell model VC-250 sonicator with a 1/8″ microtip probe, at setting #3.

Spray-Drying. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof)) can be prepared by spray drying. Spray dried iRNA can be administered to a subject or be subjected to further formulation. A pharmaceutical composition of iRNA can be prepared by spray drying a homogeneous aqueous mixture that includes a iRNA under conditions sufficient to provide a dispersible powdered composition, e.g., a pharmaceutical composition. The material for spray drying can also include one or more of: a pharmaceutically acceptable excipient, or a dispersibility-enhancing amount of a physiologically acceptable, water-soluble protein. The spray-dried product can be a dispersible powder that includes the iRNA.

Spray drying is a process that converts a liquid or slurry material to a dried particulate form. Spray drying can be used to provide powdered material for various administrative routes including inhalation. See, for example, M. Sacchetti and M. M. Van Oort in: Inhalation Aerosols: Physical and Biological Basis for Therapy, A. J. Hickey, ed. Marcel Dekkar, New York, 1996.

Spray drying can include atomizing a solution, emulsion, or suspension to form a fine mist of droplets and drying the droplets. The mist can be projected into a drying chamber (e.g., a vessel, tank, tubing, or coil) where it contacts a drying gas. The mist can include solid or liquid pore forming agents. The solvent and pore forming agents evaporate from the droplets into the drying gas to solidify the droplets, simultaneously forming pores throughout the solid. The solid (typically in a powder, particulate form) then is separated from the drying gas and collected.

Spray drying includes bringing together a highly dispersed liquid, and a sufficient volume of air (e.g., hot air) to produce evaporation and drying of the liquid droplets. The preparation to be spray dried can be any solution, course suspension, slurry, colloidal dispersion, or paste that may be atomized using the selected spray drying apparatus. Typically, the feed is sprayed into a current of warm filtered air that evaporates the solvent and conveys the dried product to a collector. The spent air is then exhausted with the solvent. Several different types of apparatus may be used to provide the desired product. For example, commercial spray dryers manufactured by Buchi Ltd. or Niro Corp. can effectively produce particles of desired size.

Spray-dried powdered particles can be approximately spherical in shape, nearly uniform in size and frequently hollow. There may be some degree of irregularity in shape depending upon the incorporated medicament and the spray drying conditions. In many instances the dispersion stability of spray-dried microspheres appears to be more effective if an inflating agent (or blowing agent) is used in their production. Particularly preferred embodiments may comprise an emulsion with an inflating agent as the disperse or continuous phase (the other phase being aqueous in nature). An inflating agent is preferably dispersed with a surfactant solution, using, for instance, a commercially available microfluidizer at a pressure of about 5000 to 15,000 psi. This process forms an emulsion, preferably stabilized by an incorporated surfactant, typically comprising submicron droplets of water immiscible blowing agent dispersed in an aqueous continuous phase. The formation of such dispersions using this and other techniques are common and well known to those in the art. The blowing agent is preferably a fluorinated compound (e.g. perfluorohexane, perfluorooctyl bromide, perfluorodecalin, perfluorobutyl ethane) which vaporizes during the spray-drying process, leaving behind generally hollow, porous aerodynamically light microspheres. As will be discussed in more detail below, other suitable blowing agents include chloroform, freons, and hydrocarbons. Nitrogen gas and carbon dioxide are also contemplated as a suitable blowing agent.

Although the perforated microstructures are preferably formed using a blowing agent as described above, it will be appreciated that, in some instances, no blowing agent is required and an aqueous dispersion of the medicament and surfactant(s) are spray dried directly. In such cases, the formulation may be amenable to process conditions (e.g., elevated temperatures) that generally lead to the formation of hollow, relatively porous microparticles. Moreover, the medicament may possess special physicochemical properties (e.g., high crystallinity, elevated melting temperature, surface activity, etc.) that make it particularly suitable for use in such techniques.

The perforated microstructures may optionally be associated with, or comprise, one or more surfactants. Moreover, miscible surfactants may optionally be combined with the suspension medium liquid phase. It will be appreciated by those skilled in the art that the use of surfactants may further increase dispersion stability, simplify formulation procedures or increase bioavailability upon administration. Of course combinations of surfactants, including the use of one or more in the liquid phase and one or more associated with the perforated microstructures are contemplated as being within the scope of the invention. By “associated with or comprise” it is meant that the structural matrix or perforated microstructure may incorporate, adsorb, absorb, be coated with or be formed by the surfactant.

Surfactants suitable for use include any compound or composition that aids in the formation and maintenance of the stabilized respiratory dispersions by forming a layer at the interface between the structural matrix and the suspension medium. The surfactant may comprise a single compound or any combination of compounds, such as in the case of co-surfactants. Particularly preferred surfactants are substantially insoluble in the propellant, nonfluorinated, and selected from the group consisting of saturated and unsaturated lipids, nonionic detergents, nonionic block copolymers, ionic surfactants, and combinations of such agents. It should be emphasized that, in addition to the aforementioned surfactants, suitable (i.e. biocompatible) fluorinated surfactants are compatible with the teachings herein and may be used to provide the desired stabilized preparations.

Lipids, including phospholipids, from both natural and synthetic sources may be used in varying concentrations to form a structural matrix. Generally, compatible lipids comprise those that have a gel to liquid crystal phase transition greater than about 40° C. Preferably, the incorporated lipids are relatively long chain (i.e. C₆-C₂₂) saturated lipids and more preferably comprise phospholipids. Exemplary phospholipids useful in the disclosed stabilized preparations comprise egg phosphatidylcholine, dilauroylphosphatidylcholine, dioleylphosphatidylcholine, dipalmitoylphosphatidyl-choline, di steroylphosphatidylcholine, short-chain phosphatidylcholines, phosphatidylethanolamine, dioleylphosphatidylethanolamine, phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, glycolipids, ganglioside GM1, sphingomyelin, phosphatidic acid, cardiolipin; lipids bearing polymer chains such as, polyethylene glycol, chitin, hyaluronic acid, or polyvinylpyrrolidone; lipids bearing sulfonated mono-, di-, and polysaccharides; fatty acids such as palmitic acid, stearic acid, and oleic acid; cholesterol, cholesterol esters, and cholesterol hemisuccinate. Due to their excellent biocompatibility characteristics, phospholipids and combinations of phospholipids and poloxamers are particularly suitable for use in the stabilized dispersions disclosed herein.

Compatible nonionic detergents comprise: sorbitan esters including sorbitan trioleate (Spans™ 85), sorbitan sesquioleate, sorbitan monooleate, sorbitan monolaurate, polyoxyethylene (20) sorbitan monolaurate, and polyoxyethylene (20) sorbitan monooleate, oleyl polyoxyethylene (2) ether, stearyl polyoxyethylene (2) ether, lauryl polyoxyethylene (4) ether, glycerol esters, and sucrose esters. Other suitable nonionic detergents can be easily identified using McCutcheon's Emulsifiers and Detergents (McPublishing Co., Glen Rock, N.J.). Preferred block copolymers include diblock and triblock copolymers of polyoxyethylene and polyoxypropylene, including poloxamer 188 (Pluronic.®. F68), poloxamer 407 (Pluronic.®. F-127), and poloxamer 338. Ionic surfactants such as sodium sulfosuccinate, and fatty acid soaps may also be utilized. In preferred embodiments, the microstructures may comprise oleic acid or its alkali salt.

In addition to the aforementioned surfactants, cationic surfactants or lipids are preferred especially in the case of delivery of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Examples of suitable cationic lipids include: DOTMA, N-[-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium-chloride; DOTAP,1,2-dioleyloxy-3-(trimethylammonio)propane; and DOTB, 1,2-dioleyl-3-(4′-trimethylammonio)butanoyl-sn-glycerol. Polycationic amino acids such as polylysine, and polyarginine are also contemplated.

For the spraying process, such spraying methods as rotary atomization, pressure atomization and two-fluid atomization can be used. Examples of the devices used in these processes include “Parubisu [phonetic rendering] Mini-Spray GA-32” and “Parubisu Spray Drier DL-41”, manufactured by Yamato Chemical Co., or “Spray Drier CL-8,” “Spray Drier L-8,” “Spray Drier FL-12,” “Spray Drier FL-16” or “Spray Drier FL-20,” manufactured by Okawara Kakoki Co., can be used for the method of spraying using rotary-disk atomizer.

While no particular restrictions are placed on the gas used to dry the sprayed material, it is recommended to use air, nitrogen gas or an inert gas. The temperature of the inlet of the gas used to dry the sprayed materials such that it does not cause heat deactivation of the sprayed material. The range of temperatures may vary between about 50° C. to about 200° C., preferably between about 50° C. and 100° C. The temperature of the outlet gas used to dry the sprayed material, may vary between about 0° C. and about 150° C., preferably between 0° C. and 90° C., and even more preferably between 0° C. and 60° C.

The spray drying is done under conditions that result in substantially amorphous powder of homogeneous constitution having a particle size that is respirable, a low moisture content and flow characteristics that allow for ready aerosolization. Preferably the particle size of the resulting powder is such that more than about 98% of the mass is in particles having a diameter of about 10 μm or less with about 90% of the mass being in particles having a diameter less than 5 μm. Alternatively, about 95% of the mass will have particles with a diameter of less than 10 μm with about 80% of the mass of the particles having a diameter of less than 5 μm.

The dispersible pharmaceutical-based dry powders that include the iRNA preparation may optionally be combined with pharmaceutical carriers or excipients which are suitable for respiratory and pulmonary administration. Such carriers may serve simply as bulking agents when it is desired to reduce the iRNA concentration in the powder which is being delivered to a patient, but may also serve to enhance the stability of the iRNA compositions and to improve the dispersibility of the powder within a powder dispersion device in order to provide more efficient and reproducible delivery of the iRNA and to improve handling characteristics of the iRNA such as flowability and consistency to facilitate manufacturing and powder filling.

Such carrier materials may be combined with the drug prior to spray drying, i.e., by adding the carrier material to the purified bulk solution. In that way, the carrier particles will be formed simultaneously with the drug particles to produce a homogeneous powder. Alternatively, the carriers may be separately prepared in a dry powder form and combined with the dry powder drug by blending. The powder carriers will usually be crystalline (to avoid water absorption), but might in some cases be amorphous or mixtures of crystalline and amorphous. The size of the carrier particles may be selected to improve the flowability of the drug powder, typically being in the range from 25 μm to 100 μm. A preferred carrier material is crystalline lactose having a size in the above-stated range.

Powders prepared by any of the above methods will be collected from the spray dryer in a conventional manner for subsequent use. For use as pharmaceuticals and other purposes, it will frequently be desirable to disrupt any agglomerates which may have formed by screening or other conventional techniques. For pharmaceutical uses, the dry powder formulations will usually be measured into a single dose, and the single dose sealed into a package. Such packages are particularly useful for dispersion in dry powder inhalers, as described in detail below. Alternatively, the powders may be packaged in multiple-dose containers.

Methods for spray drying hydrophobic and other drugs and components are described in U.S. Pat. Nos. 5,000,888; 5,026,550; 4,670,419, 4,540,602; and 4,486,435. Bloch and Speison (1983) Pharm. Acta Hely 58:14-22 teaches spray drying of hydrochlorothiazide and chlorthalidone (lipophilic drugs) and a hydrophilic adjuvant (pentaerythritol) in azeotropic solvents of dioxane-water and 2-ethoxyethanol-water. A number of Japanese Patent application Abstracts relate to spray drying of hydrophilic-hydrophobic product combinations, including JP 806766; JP 7242568; JP 7101884; JP 7101883; JP 71018982; JP 7101881; and JP 4036233. Other foreign patent publications relevant to spray drying hydrophilic-hydrophobic product combinations include FR 2594693; DE 2209477; and WO 88/07870.

Lyophilization.

An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) preparation can be made by lyophilization. Lyophilization is a freeze-drying process in which water is sublimed from the composition after it is frozen. The particular advantage associated with the lyophilization process is that biologicals and pharmaceuticals that are relatively unstable in an aqueous solution can be dried without elevated temperatures (thereby eliminating the adverse thermal effects), and then stored in a dry state where there are few stability problems. With respect to the instant invention such techniques are particularly compatible with the incorporation of nucleic acids in perforated microstructures without compromising physiological activity. Methods for providing lyophilized particulates are known to those of skill in the art and it would clearly not require undue experimentation to provide dispersion compatible microstructures in accordance with the teachings herein. Accordingly, to the extent that lyophilization processes may be used to provide microstructures having the desired porosity and size, they are conformance with the teachings herein and are expressly contemplated as being within the scope of the instant invention.

Targeting

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNAs. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention.

In some embodiments, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is targeted to a particular cell. For example, a liposome or particle or other structure that includes a iRNA can also include a targeting moiety that recognizes a specific molecule on a target cell. The targeting moiety can be a molecule with a specific affinity for a target cell. Targeting moieties can include antibodies directed against a protein found on the surface of a target cell, or the ligand or a receptor-binding portion of a ligand for a molecule found on the surface of a target cell. For example, the targeting moiety can recognize a cancer-specific antigen (e.g., CA15-3, CA19-9, CEA, or HER^(2/)neu.) or a viral antigen, thus delivering the iRNA to a cancer cell or a virus-infected cell. Exemplary targeting moieties include antibodies (such as IgM, IgG, IgA, IgD, and the like, or a functional portions thereof), ligands for cell surface receptors (e.g., ectodomains thereof).

Table 3 provides a number of antigens which can be used to target selected cells.

TABLE 3 ANTIGEN Exemplary tumor tissue CEA (carcinoembryonic antigen) colon, breast, lung PSA (prostate specific antigen) prostate cancer CA-125 ovarian cancer CA 15-3 breast cancer CA 19-9 breast cancer HER2/neu breast cancer α-feto protein testicular cancer, hepatic cancer β-HCG (human chorionic testicular cancer, gonadotropin) choriocarcinoma MUC-1 breast cancer Estrogen receptor breast cancer, uterine cancer Progesterone receptor breast cancer, uterine cancer EGFr (epidermal growth bladder cancer factor receptor)

In one embodiment, the targeting moiety is attached to a liposome. For example, U.S. Pat. No. 6,245,427 describes a method for targeting a liposome using a protein or peptide. In another example, a cationic lipid component of the liposome is derivatized with a targeting moiety.

For example, WO 96/37194 describes converting N-glutaryldioleoylphosphatidyl ethanolamine to a N-hydroxysuccinimide activated ester. The product was then coupled to an RGD peptide.

Genes and Diseases

In one aspect, the invention features, a method of treating a subject at risk for or afflicted with unwanted cell proliferation, e.g., malignant or nonmalignant cell proliferation.

The method includes:

providing an iRNA agent, e.g., an sRNA or iRNA agent described herein, e.g., an iRNA having a structure described herein, where the iRNA is homologous to and can silence, e.g., by cleavage, a gene which promotes unwanted cell proliferation;

administering an iRNA agent, e.g., an sRNA or iRNA agent described herein to a subject, preferably a human subject,

thereby treating the subject.

In a preferred embodiment the gene is a growth factor or growth factor receptor gene, a kinase, e.g., a protein tyrosine, serine or threonine kinase gene, an adaptor protein gene, a gene encoding a G protein superfamily molecule, or a gene encoding a transcription factor.

In a preferred embodiment the iRNA agent silences the PDGF beta gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PDGF beta expression, e.g., testicular and lung cancers.

In another preferred embodiment the iRNA agent silences the Erb-B gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erb-B expression, e.g., breast cancer

In a preferred embodiment the iRNA agent silences the Src gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Src expression, e.g., colon cancers.

In a preferred embodiment the iRNA agent silences the CRK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted CRK expression, e.g., colon and lung cancers.

In a preferred embodiment the iRNA agent silences the GRB2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted GRB2 expression, e.g., squamous cell carcinoma.

In another preferred embodiment the iRNA agent silences the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., pancreatic, colon and lung cancers, and chronic leukemia.

In another preferred embodiment the iRNA agent silences the MEKK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MEKK expression, e.g., squamous cell carcinoma, melanoma or leukemia.

In another preferred embodiment the iRNA agent silences the JNK gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted INK expression, e.g., pancreatic or breast cancers.

In a preferred embodiment the iRNA agent silences the RAF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAF expression, e.g., lung cancer or leukemia.

In a preferred embodiment the iRNA agent silences the Erk1/2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Erk1/2 expression, e.g., lung cancer.

In another preferred embodiment the iRNA agent silences the PCNA(p21) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PCNA expression, e.g., lung cancer.

In a preferred embodiment the iRNA agent silences the MYB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MYB expression, e.g., colon cancer or chronic myelogenous leukemia.

In a preferred embodiment the iRNA agent silences the c-MYC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MYC expression, e.g., Burkitt's lymphoma or neuroblastoma.

In another preferred embodiment the iRNA agent silences the JUN gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted JUN expression, e.g., ovarian, prostate or breast cancers.

In another preferred embodiment the iRNA agent silences the FOS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted FOS expression, e.g., skin or prostate cancers.

In a preferred embodiment the iRNA agent silences the BCL-2 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCL-2 expression, e.g., lung or prostate cancers or Non-Hodgkin lymphoma.

In a preferred embodiment the iRNA agent silences the Cyclin D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin D expression, e.g., esophageal and colon cancers.

In a preferred embodiment the iRNA agent silences the VEGF gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted VEGF expression, e.g., esophageal and colon cancers.

In a preferred embodiment the iRNA agent silences the EGFR gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EGFR expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences the Cyclin A gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin A expression, e.g., lung and cervical cancers.

In another preferred embodiment the iRNA agent silences the Cyclin E gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Cyclin E expression, e.g., lung and breast cancers.

In another preferred embodiment the iRNA agent silences the WNT-1 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted WNT-1 expression, e.g., basal cell carcinoma.

In another preferred embodiment the iRNA agent silences the beta-catenin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted beta-catenin expression, e.g., adenocarcinoma or hepatocellular carcinoma.

In another preferred embodiment the iRNA agent silences the c-MET gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted c-MET expression, e.g., hepatocellular carcinoma.

In another preferred embodiment the iRNA agent silences the PKC gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PKC expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the NFKB gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted NFKB expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences the STAT3 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted STAT3 expression, e.g., prostate cancer.

In another preferred embodiment the iRNA agent silences the survivin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted survivin expression, e.g., cervical or pancreatic cancers.

In another preferred embodiment the iRNA agent silences the Her2/Neu gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Her2/Neu expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences the topoisomerase I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase I expression, e.g., ovarian and colon cancers.

In a preferred embodiment the iRNA agent silences the topoisomerase II alpha gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted topoisomerase II expression, e.g., breast and colon cancers.

In a preferred embodiment the iRNA agent silences mutations in the p73 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p73 expression, e.g., colorectal adenocarcinoma.

In a preferred embodiment the iRNA agent silences mutations in the p21(WAF1/CIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p21(WAF1/CIP1) expression, e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in the p27(KIP1) gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p27(KIP1) expression, e.g., liver cancer.

In a preferred embodiment the iRNA agent silences mutations in the PPM1D gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PPM1D expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences mutations in the RAS gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted RAS expression, e.g., breast cancer.

In another preferred embodiment the iRNA agent silences mutations in the caveolin I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted caveolin I expression, e.g., esophageal squamous cell carcinoma.

In another preferred embodiment the iRNA agent silences mutations in the MIB I gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MIB I expression, e.g., male breast carcinoma (MBC).

In another preferred embodiment the iRNA agent silences mutations in the MTAI gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MTAI expression, e.g., ovarian carcinoma.

In another preferred embodiment the iRNA agent silences mutations in the M68 gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted M68 expression, e.g., human adenocarcinomas of the esophagus, stomach, colon, and rectum.

In preferred embodiments the iRNA agent silences mutations in tumor suppressor genes, and thus can be used as a method to promote apoptotic activity in combination with chemotherapeutics.

In a preferred embodiment the iRNA agent silences mutations in the p53 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted p53 expression, e.g., gall bladder, pancreatic and lung cancers.

In a preferred embodiment the iRNA agent silences mutations in the p53 family member DN-p63, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted DN-p63 expression, e.g., squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the pRb tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted pRb expression, e.g., oral squamous cell carcinoma

In a preferred embodiment the iRNA agent silences mutations in the APC1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted APC1 expression, e.g., colon cancer.

In a preferred embodiment the iRNA agent silences mutations in the BRCA1 tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BRCA1 expression, e.g., breast cancer.

In a preferred embodiment the iRNA agent silences mutations in the PTEN tumor suppressor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PTEN expression, e.g., hamartomas, gliomas, and prostate and endometrial cancers.

In a preferred embodiment the iRNA agent silences MLL fusion genes, e.g., MLL-AF9, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted MLL fusion gene expression, e.g., acute leukemias.

In another preferred embodiment the iRNA agent silences the BCR/ABL fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted BCR/ABL fusion gene expression, e.g., acute and chronic leukemias.

In another preferred embodiment the iRNA agent silences the TEL/AML1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TEL/AML1 fusion gene expression, e.g., childhood acute leukemia.

In another preferred embodiment the iRNA agent silences the EWS/FLI1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted EWS/FLI1 fusion gene expression, e.g., Ewing Sarcoma.

In another preferred embodiment the iRNA agent silences the TLS/FUS1 fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted TLS/FUS1 fusion gene expression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the PAX3/FKHR fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted PAX3/FKHR fusion gene expression, e.g., Myxoid liposarcoma.

In another preferred embodiment the iRNA agent silences the AML1/ETO fusion gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted AML1/ETO fusion gene expression, e.g., acute leukemia.

In another aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder that may benefit by angiogenesis inhibition e.g., cancer. The method includes:

providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates angiogenesis;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the iRNA agent silences the alpha v-integrin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted alpha V integrin, e.g., brain tumors or tumors of epithelial origin.

In a preferred embodiment the iRNA agent silences the Flt-1 receptor gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted Flt-1 receptors, eg. Cancer and rheumatoid arthritis.

In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization.

In a preferred embodiment the iRNA agent silences the tubulin gene, and thus can be used to treat a subject having or at risk for a disorder characterized by unwanted tubulin, eg. Cancer and retinal neovascularization.

In another aspect, the invention features a method of treating a subject infected with a virus or at risk for or afflicted with a disorder or disease associated with a viral infection.

The method includes:

providing an iRNA agent, e.g., and iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a viral gene of a cellular gene which mediates viral function, e.g., entry or growth;

administering the iRNA agent to a subject, preferably a human subject,

thereby treating the subject.

Thus, the invention provides for a method of treating patients infected by the Human Papilloma Virus (HPV) or at risk for or afflicted with a disorder mediated by HPV, e.g, cervical cancer. HPV is linked to 95% of cervical carcinomas and thus an antiviral therapy is an attractive method to treat these cancers and other symptoms of viral infection.

In a preferred embodiment, the expression of a HPV gene is reduced. In another preferred embodiment, the HPV gene is one of the group of E2, E6, or E7.

In a preferred embodiment the expression of a human gene that is required for HPV replication is reduced.

The invention also includes a method of treating patients infected by the Human Immunodeficiency Virus (HIV) or at risk for or afflicted with a disorder mediated by HIV, e.g., Acquired Immune Deficiency Syndrome (AIDS).

In a preferred embodiment, the expression of a HIV gene is reduced. In another preferred embodiment, the HIV gene is CCRS, Gag, or Rev.

In a preferred embodiment the expression of a human gene that is required for HIV replication is reduced. In another preferred embodiment, the gene is CD4 or Tsg101.

The invention also includes a method for treating patients infected by the Hepatitis B Virus (HBV) or at risk for or afflicted with a disorder mediated by HBV, e.g., cirrhosis and heptocellular carcinoma.

In a preferred embodiment, the expression of a HBV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of the tail region of the HBV core protein, the pre-cregious (pre-c) region, or the cregious (c) region. In another preferred embodiment, a targeted HBV-RNA sequence is comprised of the poly(A) tail.

In preferred embodiment the expression of a human gene that is required for HBV replication is reduced.

The invention also provides for a method of treating patients infected by the Hepatitis A Virus (HAV), or at risk for or afflicted with a disorder mediated by HAV.

In a preferred embodiment the expression of a human gene that is required for HAV replication is reduced.

The present invention provides for a method of treating patients infected by the Hepatitis C Virus (HCV), or at risk for or afflicted with a disorder mediated by HCV, e.g., cirrhosis

In a preferred embodiment, the expression of a HCV gene is reduced.

In another preferred embodiment the expression of a human gene that is required for

HCV replication is reduced.

The present invention also provides for a method of treating patients infected by the any of the group of Hepatitis Viral strains comprising hepatitis D, E, F, G, or H, or patients at risk for or afflicted with a disorder mediated by any of these strains of hepatitis.

In a preferred embodiment, the expression of a Hepatitis, D, E, F, G, or H gene is reduced.

In another preferred embodiment the expression of a human gene that is required for hepatitis D, E, F, G or H replication is reduced.

Methods of the invention also provide for treating patients infected by the Respiratory Syncytial Virus (RSV) or at risk for or afflicted with a disorder mediated by RSV, e.g, lower respiratory tract infection in infants and childhood asthma, pneumonia and other complications, e.g., in the elderly.

In a preferred embodiment, the expression of a RSV gene is reduced. In another preferred embodiment, the targeted HBV gene encodes one of the group of genes N, L, or P.

In a preferred embodiment the expression of a human gene that is required for RSV replication is reduced.

Methods of the invention provide for treating patients infected by the Herpes Simplex Virus (HSV) or at risk for or afflicted with a disorder mediated by HSV, e.g, genital herpes and cold sores as well as life-threatening or sight-impairing disease mainly in immunocompromised patients.

In a preferred embodiment, the expression of a HSV gene is reduced. In another preferred embodiment, the targeted HSV gene encodes DNA polymerase or the helicase-primase.

In a preferred embodiment the expression of a human gene that is required for HSV replication is reduced.

The invention also provides a method for treating patients infected by the herpes Cytomegalovirus (CMV) or at risk for or afflicted with a disorder mediated by CMV, e.g., congenital virus infections and morbidity in immunocompromised patients.

In a preferred embodiment, the expression of a CMV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for CMV replication is reduced.

Methods of the invention also provide for a method of treating patients infected by the herpes Epstein Barr Virus (EBV) or at risk for or afflicted with a disorder mediated by EBV, e.g., NK/T-cell lymphoma, non-Hodgkin lymphoma, and Hodgkin disease.

In a preferred embodiment, the expression of a EBV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for EBV replication is reduced.

Methods of the invention also provide for treating patients infected by Kaposi's Sarcoma-associated Herpes Virus (KSHV), also called human herpesvirus 8, or patients at risk for or afflicted with a disorder mediated by KSHV, e.g., Kaposi's sarcoma, multicentric Castleman's disease and AIDS-associated primary effusion lymphoma.

In a preferred embodiment, the expression of a KSHV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for KSHV replication is reduced.

The invention also includes a method for treating patients infected by the JC Virus (JCV) or a disease or disorder associated with this virus, e.g., progressive multifocal leukoencephalopathy (PML).

In a preferred embodiment, the expression of a JCV gene is reduced.

In preferred embodiment the expression of a human gene that is required for JCV replication is reduced.

Methods of the invention also provide for treating patients infected by the myxovirus or at risk for or afflicted with a disorder mediated by myxovirus, e.g., influenza.

In a preferred embodiment, the expression of a myxovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for myxovirus replication is reduced.

Methods of the invention also provide for treating patients infected by the rhinovirus or at risk for of afflicted with a disorder mediated by rhinovirus, e.g., the common cold.

In a preferred embodiment, the expression of a rhinovirus gene is reduced.

In preferred embodiment the expression of a human gene that is required for rhinovirus replication is reduced.

Methods of the invention also provide for treating patients infected by the coronavirus or at risk for of afflicted with a disorder mediated by coronavirus, e.g., the common cold.

In a preferred embodiment, the expression of a coronavirus gene is reduced.

In preferred embodiment the expression of a human gene that is required for coronavirus replication is reduced.

Methods of the invention also provide for treating patients infected by the flavivirus West Nile or at risk for or afflicted with a disorder mediated by West Nile Virus.

In a preferred embodiment, the expression of a West Nile Virus gene is reduced. In another preferred embodiment, the West Nile Virus gene is one of the group comprising E, NS3, or NS5.

In a preferred embodiment the expression of a human gene that is required for West Nile Virus replication is reduced.

Methods of the invention also provide for treating patients infected by the St. Louis Encephalitis flavivirus, or at risk for or afflicted with a disease or disorder associated with this virus, e.g., viral haemorrhagic fever or neurological disease.

In a preferred embodiment, the expression of a St. Louis Encephalitis gene is reduced.

In a preferred embodiment the expression of a human gene that is required for St. Louis Encephalitis virus replication is reduced.

Methods of the invention also provide for treating patients infected by the Tick-borne encephalitis flavivirus, or at risk for or afflicted with a disorder mediated by Tick-borne encephalitis virus, e.g., viral haemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Tick-borne encephalitis virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Tick-borne encephalitis virus replication is reduced.

Methods of the invention also provide for methods of treating patients infected by the Murray Valley encephalitis flavivirus, which commonly results in viral haemorrhagic fever and neurological disease.

In a preferred embodiment, the expression of a Murray Valley encephalitis virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Murray Valley encephalitis virus replication is reduced.

The invention also includes methods for treating patients infected by the dengue flavivirus, or a disease or disorder associated with this virus, e.g., dengue haemorrhagic fever.

In a preferred embodiment, the expression of a dengue virus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for dengue virus replication is reduced.

Methods of the invention also provide for treating patients infected by the Simian Virus 40 (SV40) or at risk for or afflicted with a disorder mediated by SV40, e.g., tumorigenesis.

In a preferred embodiment, the expression of a SV40 gene is reduced.

In a preferred embodiment the expression of a human gene that is required for SV40 replication is reduced.

The invention also includes methods for treating patients infected by the Human T Cell Lymphotropic Virus (HTLV), or a disease or disorder associated with this virus, e.g., leukemia and myelopathy.

In a preferred embodiment, the expression of a HTLV gene is reduced. In another preferred embodiment the HTLV1 gene is the Tax transcriptional activator.

In a preferred embodiment the expression of a human gene that is required for HTLV replication is reduced.

Methods of the invention also provide for treating patients infected by the Moloney-Murine Leukemia Virus (Mo-MuLV) or at risk for or afflicted with a disorder mediated by Mo-MuLV, e.g., T-cell leukemia.

In a preferred embodiment, the expression of a Mo-MuLV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mo-MuLV replication is reduced.

Methods of the invention also provide for treating patients infected by the encephalomyocarditis virus (EMCV) or at risk for or afflicted with a disorder mediated by EMCV, e.g. myocarditis. EMCV leads to myocarditis in mice and pigs and is capable of infecting human myocardial cells. This virus is therefore a concern for patients undergoing xenotransplantation.

In a preferred embodiment, the expression of a EMCV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for EMCV replication is reduced.

The invention also includes a method for treating patients infected by the measles virus (MV) or at risk for or afflicted with a disorder mediated by MV, e.g. measles.

In a preferred embodiment, the expression of a MV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for MV replication is reduced.

The invention also includes a method for treating patients infected by the Vericella zoster virus (VZV) or at risk for or afflicted with a disorder mediated by VZV, e.g. chicken pox or shingles (also called zoster).

In a preferred embodiment, the expression of a VZV gene is reduced.

In a preferred embodiment the expression of a human gene that is required for VZV replication is reduced.

The invention also includes a method for treating patients infected by an adenovirus or at risk for or afflicted with a disorder mediated by an adenovirus, e.g. respiratory tract infection.

In a preferred embodiment, the expression of an adenovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for adenovirus replication is reduced.

The invention includes a method for treating patients infected by a yellow fever virus

(YFV) or at risk for or afflicted with a disorder mediated by a YFV, e.g. respiratory tract infection.

In a preferred embodiment, the expression of a YFV gene is reduced. In another preferred embodiment, the preferred gene is one of a group that includes the E, NS2A, or NS3 genes.

In a preferred embodiment the expression of a human gene that is required for YFV replication is reduced.

Methods of the invention also provide for treating patients infected by the poliovirus or at risk for or afflicted with a disorder mediated by poliovirus, e.g., polio.

In a preferred embodiment, the expression of a poliovirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for poliovirus replication is reduced.

Methods of the invention also provide for treating patients infected by a poxvirus or at risk for or afflicted with a disorder mediated by a poxvirus, e.g., smallpox

In a preferred embodiment, the expression of a poxvirus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for poxvirus replication is reduced.

In another, aspect the invention features methods of treating a subject infected with a pathogen, e.g., a bacterial, amoebic, parasitic, or fungal pathogen. The method includes:

providing a iRNA agent, e.g., a siRNA having a structure described herein, where siRNA is homologous to and can silence, e.g., by cleavage of a pathogen gene;

administering the iRNA agent to a subject, prefereably a human subject,

thereby treating the subject.

The target gene can be one involved in growth, cell wall synthesis, protein synthesis, transcription, energy metabolism, e.g., the Krebs cycle, or toxin production.

Thus, the present invention provides for a method of treating patients infected by a plasmodium that causes malaria.

In a preferred embodiment, the expression of a plasmodium gene is reduced. In another preferred embodiment, the gene is apical membrane antigen 1 (AMA1).

In a preferred embodiment the expression of a human gene that is required for plasmodium replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium ulcerans, or a disease or disorder associated with this pathogen, e.g. Buruli ulcers.

In a preferred embodiment, the expression of a Mycobacterium ulcerans gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium ulcerans replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium tuberculosis, or a disease or disorder associated with this pathogen, e.g. tuberculosis.

In a preferred embodiment, the expression of a Mycobacterium tuberculosis gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium tuberculosis replication is reduced.

The invention also includes methods for treating patients infected by the Mycobacterium leprae, or a disease or disorder associated with this pathogen, e.g. leprosy.

In a preferred embodiment, the expression of a Mycobacterium leprae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycobacterium leprae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Staphylococcus aureus, or a disease or disorder associated with this pathogen, e.g. infections of the skin and muscous membranes.

In a preferred embodiment, the expression of a Staphylococcus aureus gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Staphylococcus aureus replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Streptococcus pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection.

In a preferred embodiment, the expression of a Streptococcus pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Streptococcus pneumoniae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Streptococcus pyogenes, or a disease or disorder associated with this pathogen, e.g. Strep throat or Scarlet fever.

In a preferred embodiment, the expression of a Streptococcus pyogenes gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Streptococcus pyogenes replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Chlamydia pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection

In a preferred embodiment, the expression of a Chlamydia pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Chlamydia pneumoniae replication is reduced.

The invention also includes methods for treating patients infected by the bacteria Mycoplasma pneumoniae, or a disease or disorder associated with this pathogen, e.g. pneumonia or childhood lower respiratory tract infection

In a preferred embodiment, the expression of a Mycoplasma pneumoniae gene is reduced.

In a preferred embodiment the expression of a human gene that is required for Mycoplasma pneumoniae replication is reduced.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a disease or disorder characterized by an unwanted immune response, e.g., an inflammatory disease or disorder, or an autoimmune disease or disorder.

The method includes:

providing an iRNA agent, e.g., an iRNA agent having a structure described herein, which iRNA agent is homologous to and can silence, e.g., by cleavage, a gene which mediates an unwanted immune response;

administering the iRNA agent to a subject,

thereby treating the subject.

In a preferred embodiment the disease or disorder is an ischemia or reperfusion injury, e.g., ischemia or reperfusion injury associated with acute myocardial infarction, unstable angina, cardiopulmonary bypass, surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty, the response to a transplantated organ or tissue, e.g., transplanted cardiac or vascular tissue; or thrombolysis.

In a preferred embodiment the disease or disorder is restenosis, e.g., restenosis associated with surgical intervention e.g., angioplasty, e.g., percutaneous transluminal coronary angioplasty.

In a prefered embodiment the disease or disorder is Inflammatory Bowel Disease, e.g., Crohn Disease or Ulcerative Colitis.

In a prefered embodiment the disease or disorder is inflammation associated with an infection or injury.

In a prefered embodiment the disease or disorder is asthma, lupus, multiple sclerosis, diabetes, e.g., type II diabetes, arthritis, e.g., rheumatoid or psoriatic.

In particularly preferred embodiments the iRNA agent silences an integrin or co-ligand thereof, e.g., VLA4, VCAM, ICAM.

In particularly preferred embodiments the iRNA agent silences a selectin or co-ligand thereof, e.g., P-selectin, E-selectin (ELAM), I-selectin, P-selectin glycoprotein-1 (PSGL-1).

In particularly preferred embodiments the iRNA agent silences a component of the complement system, e.g., C3, C5, C3aR, C5aR, C3 convertase, C5 convertase.

In particularly preferred embodiments the iRNA agent silences a chemokine or receptor thereof, e.g., TNFI, TNFJ, IL-1I, IL-1J, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-6, IL-8, TNFRI, TNFRII, IgE, SCYA11, CCR^(3.)

In other embodiments the iRNA agent silences GCSF, Gro1, Gro2, Gro3, PF4, MIG, Pro-Platelet Basic Protein (PPBP), MIP-1I, MIP-1J, RANTES, MCP-1, MCP-2, MCP-3, CMBKR1, CMBKR2, CMBKR3, CMBKR5, AIF-1, I-309.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with acute pain or chronic pain. The method includes:

providing an iRNA agent, which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates the processing of pain;

administering the iRNA to a subject,

thereby treating the subject.

In particularly preferred embodiments the iRNA agent silences a component of an ion channel.

In particularly preferred embodiments the iRNA agent silences a neurotransmitter receptor or ligand.

In one aspect, the invention features, a method of treating a subject, e.g., a human, at risk for or afflicted with a neurological disease or disorder. The method includes:

providing an iRNA agent which iRNA is homologous to and can silence, e.g., by cleavage, a gene which mediates a neurological disease or disorder;

administering the to a subject,

thereby treating the subject.

In a prefered embodiment the disease or disorder is Alzheimer Disease or Parkinson Disease.

In particularly preferred embodiments the iRNA agent silences an amyloid-family gene, e.g., APP; a presenilin gene, e.g., PSEN1 and PSEN2, or I-synuclein.

In a preferred embodiment the disease or disorder is a neurodegenerative trinucleotide repeat disorder, e.g., Huntington disease, dentatorubral pallidoluysian atrophy or a spinocerebellar ataxia, e.g., SCA1, SCA2, SCA3 (Machado-Joseph disease), SCAT or SCAB.

In particularly preferred embodiments the iRNA agent silences HD, DRPLA, SCA1, SCA2, MJD1, CACNL1A4, SCAT, SCAB.

The loss of heterozygosity (LOH) can result in hemizygosity for sequence, e.g., genes, in the area of LOH. This can result in a significant genetic difference between normal and disease-state cells, e.g., cancer cells, and provides a useful difference between normal and disease-state cells, e.g., cancer cells. This difference can arise because a gene or other sequence is heterozygous in euploid cells but is hemizygous in cells having LOH. The regions of LOH will often include a gene, the loss of which promotes unwanted proliferation, e.g., a tumor suppressor gene, and other sequences including, e.g., other genes, in some cases a gene which is essential for normal function, e.g., growth. Methods of the invention rely, in part, on the specific cleavage or silencing of one allele of an essential gene with an iRNA agent of the invention. The iRNA agent is selected such that it targets the single allele of the essential gene found in the cells having LOH but does not silence the other allele, which is present in cells which do not show LOH. In essence, it discriminates between the two alleles, preferentially silencing the selected allele. In essence polymorphisms, e.g., SNPs of essential genes that are affected by LOH, are used as a target for a disorder characterized by cells having LOH, e.g., cancer cells having LOH.

E.g., one of ordinary skill in the art can identify essential genes which are in proximity to tumor suppressor genes, and which are within a LOH region which includes the tumor suppressor gene. The gene encoding the large subunit of human RNA polymerase II, POLR²A, a gene located in close proximity to the tumor suppressor gene p53, is such a gene. It frequently occurs within a region of LOH in cancer cells. Other genes that occur within LOH regions and are lost in many cancer cell types include the group comprising replication protein A 70-kDa subunit, replication protein A 32-kD, ribonucleotide reductase, thymidilate synthase, TATA associated factor 2H, ribosomal protein S14, eukaryotic initiation factor 5A, alanyl tRNA synthetase, cysteinyl tRNA synthetase, NaK ATPase, alpha-1 subunit, and transferrin receptor.

Accordingly, the invention features, a method of treating a disorder characterized by LOH, e.g., cancer. The method includes:

optionally, determining the genotype of the allele of a gene in the region of LOH and preferably determining the genotype of both alleles of the gene in a normal cell;

providing an iRNA agent which preferentially cleaves or silences the allele found in the LOH cells;

administerning the iRNA to the subject,

thereby treating the disorder.

The invention also includes a iRNA agent disclosed herein, e.g, an iRNA agent which can preferentially silence, e.g., cleave, one allele of a polymorphic gene

In another aspect, the invention provides a method of cleaving or silencing more than one gene with an iRNA agent. In these embodiments the iRNA agent is selected so that it has sufficient homology to a sequence found in more than one gene. For example, the sequence AAGCTGGCCCTGGACATGGAGAT (SEQ ID NO:6736) is conserved between mouse lamin B1, lamin B2, keratin complex 2-gene 1 and lamin A/C. Thus an iRNA agent targeted to this sequence would effectively silence the entire collection of genes.

The invention also includes an iRNA agent disclosed herein, which can silence more than one gene.

Route of Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes a iRNA can be delivered to a subject by a variety of routes. Exemplary routes include: intravenous, topical, rectal, anal, vaginal, nasal, pulmonary, ocular.

The iRNA molecules of the invention can be incorporated into pharmaceutical compositions suitable for administration. Such compositions typically include one or more species of iRNA and a pharmaceutically acceptable carrier. As used herein the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active compound, use thereof in the compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions.

The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic, vaginal, rectal, intranasal, transdermal), oral or parenteral. Parenteral administration includes intravenous drip, subcutaneous, intraperitoneal or intramuscular injection, or intrathecal or intraventricular administration.

The route and site of administration may be chosen to enhance targeting. For example, to target muscle cells, intramuscular injection into the muscles of interest would be a logical choice. Lung cells might be targeted by administering the iRNA in aerosol form. The vascular endothelial cells could be targeted by coating a balloon catheter with the iRNA and mechanically introducing the DNA.

Formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable. Coated condoms, gloves and the like may also be useful.

Compositions for oral administration include powders or granules, suspensions or solutions in water, syrups, elixirs or non-aqueous media, tablets, capsules, lozenges, or troches. In the case of tablets, carriers that can be used include lactose, sodium citrate and salts of phosphoric acid. Various disintegrants such as starch, and lubricating agents such as magnesium stearate, sodium lauryl sulfate and talc, are commonly used in tablets. For oral administration in capsule form, useful diluents are lactose and high molecular weight polyethylene glycols. When aqueous suspensions are required for oral use, the nucleic acid compositions can be combined with emulsifying and suspending agents. If desired, certain sweetening and/or flavoring agents can be added.

Compositions for intrathecal or intraventricular administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives.

Formulations for parenteral administration may include sterile aqueous solutions which may also contain buffers, diluents and other suitable additives. Intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir. For intravenous use, the total concentration of solutes should be controlled to render the preparation isotonic.

For ocular administration, ointments or droppable liquids may be delivered by ocular delivery systems known to the art such as applicators or eye droppers. Such compositions can include mucomimetics such as hyaluronic acid, chondroitin sulfate, hydroxypropyl methylcellulose or poly(vinyl alcohol), preservatives such as sorbic acid, EDTA or benzylchronium chloride, and the usual quantities of diluents and/or carriers.

Topical Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. In a preferred embodiment, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) is delivered to a subject via topical administration. “Topical administration” refers to the delivery to a subject by contacting the formulation directly to a surface of the subject. The most common form of topical delivery is to the skin, but a composition disclosed herein can also be directly applied to other surfaces of the body, e.g., to the eye, a mucous membrane, to surfaces of a body cavity or to an internal surface. As mentioned above, the most common topical delivery is to the skin. The term encompasses several routes of administration including, but not limited to, topical and transdermal. These modes of administration typically include penetration of the skin's permeability barrier and efficient delivery to the target tissue or stratum. Topical administration can be used as a means to penetrate the epidermis and dermis and ultimately achieve systemic delivery of the composition. Topical administration can also be used as a means to selectively deliver oligonucleotides to the epidermis or dermis of a subject, or to specific strata thereof, or to an underlying tissue.

The term “skin,” as used herein, refers to the epidermis and/or dermis of an animal. Mammalian skin consists of two major, distinct layers. The outer layer of the skin is called the epidermis. The epidermis is comprised of the stratum corneum, the stratum granulosum, the stratum spinosum, and the stratum basale, with the stratum corneum being at the surface of the skin and the stratum basale being the deepest portion of the epidermis. The epidermis is between 50 μm and 0.2 mm thick, depending on its location on the body.

Beneath the epidermis is the dermis, which is significantly thicker than the epidermis. The dermis is primarily composed of collagen in the form of fibrous bundles. The collagenous bundles provide support for, inter alia, blood vessels, lymph capillaries, glands, nerve endings and immunologically active cells.

One of the major functions of the skin as an organ is to regulate the entry of substances into the body. The principal permeability barrier of the skin is provided by the stratum corneum, which is formed from many layers of cells in various states of differentiation. The spaces between cells in the stratum corneum is filled with different lipids arranged in lattice-like formations that provide seals to further enhance the skins permeability barrier.

The permeability barrier provided by the skin is such that it is largely impermeable to molecules having molecular weight greater than about 750 Da. For larger molecules to cross the skin's permeability barrier, mechanisms other than normal osmosis must be used.

Several factors determine the permeability of the skin to administered agents. These factors include the characteristics of the treated skin, the characteristics of the delivery agent, interactions between both the drug and delivery agent and the drug and skin, the dosage of the drug applied, the form of treatment, and the post treatment regimen. To selectively target the epidermis and dermis, it is sometimes possible to formulate a composition that comprises one or more penetration enhancers that will enable penetration of the drug to a preselected stratum.

Transdermal delivery is a valuable route for the administration of lipid soluble therapeutics. The dermis is more permeable than the epidermis and therefore absorption is much more rapid through abraded, burned or denuded skin. Inflammation and other physiologic conditions that increase blood flow to the skin also enhance transdermal adsorption. Absorption via this route may be enhanced by the use of an oily vehicle (inunction) or through the use of one or more penetration enhancers. Other effective ways to deliver a composition disclosed herein via the transdermal route include hydration of the skin and the use of controlled release topical patches. The transdermal route provides a potentially effective means to deliver a composition disclosed herein for systemic and/or local therapy.

In addition, iontophoresis (transfer of ionic solutes through biological membranes under the influence of an electric field) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 163), phonophoresis or sonophoresis (use of ultrasound to enhance the absorption of various therapeutic agents across biological membranes, notably the skin and the cornea) (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 166), and optimization of vehicle characteristics relative to dose position and retention at the site of administration (Lee et al., Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 168) may be useful methods for enhancing the transport of topically applied compositions across skin and mucosal sites.

The compositions and methods provided may also be used to examine the function of various proteins and genes in vitro in cultured or preserved dermal tissues and in animals. The invention can be thus applied to examine the function of any gene. The methods of the invention can also be used therapeutically or prophylactically. For example, for the treatment of animals that are known or suspected to suffer from diseases such as psoriasis, lichen planus, toxic epidermal necrolysis, ertythema multiforme, basal cell carcinoma, squamous cell carcinoma, malignant melanoma, Paget's disease, Kaposi's sarcoma, pulmonary fibrosis, Lyme disease and viral, fungal and bacterial infections of the skin.

Pulmonary Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. A composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be administered to a subject by pulmonary delivery. Pulmonary delivery compositions can be delivered by inhalation by the patient of a dispersion so that the composition, preferably iRNA, within the dispersion can reach the lung where it can be readily absorbed through the alveolar region directly into blood circulation. Pulmonary delivery can be effective both for systemic delivery and for localized delivery to treat diseases of the lungs.

Pulmonary delivery can be achieved by different approaches, including the use of nebulized, aerosolized, micellular and dry powder-based formulations. Delivery can be achieved with liquid nebulizers, aerosol-based inhalers, and dry powder dispersion devices. Metered-dose devices are preferred. One of the benefits of using an atomizer or inhaler is that the potential for contamination is minimized because the devices are self contained. Dry powder dispersion devices, for example, deliver drugs that may be readily formulated as dry powders. A iRNA composition may be stably stored as lyophilized or spray-dried powders by itself or in combination with suitable powder carriers. The delivery of a composition for inhalation can be mediated by a dosing timing element which can include a timer, a dose counter, time measuring device, or a time indicator which when incorporated into the device enables dose tracking, compliance monitoring, and/or dose triggering to a patient during administration of the aerosol medicament.

The term “powder” means a composition that consists of finely dispersed solid particles that are free flowing and capable of being readily dispersed in an inhalation device and subsequently inhaled by a subject so that the particles reach the lungs to permit penetration into the alveoli. Thus, the powder is said to be “respirable.” Preferably the average particle size is less than about 10 μm in diameter preferably with a relatively uniform spheroidal shape distribution. More preferably the diameter is less than about 7.5 μm and most preferably less than about 5.0. Usually the particle size distribution is between about 0.1 μm and about 5 μm in diameter, particularly about 0.3 μm to about 5 μm.

The term “dry” means that the composition has a moisture content below about 10% by weight (% w) water, usually below about 5% w and preferably less it than about 3% w. A dry composition can be such that the particles are readily dispersible in an inhalation device to form an aerosol.

The term “therapeutically effective amount” is the amount present in the composition that is needed to provide the desired level of drug in the subject to be treated to give the anticipated physiological response.

The term “physiologically effective amount” is that amount delivered to a subject to give the desired palliative or curative effect.

The term “pharmaceutically acceptable carrier” means that the carrier can be taken into the lungs with no significant adverse toxicological effects on the lungs.

The types of pharmaceutical excipients that are useful as carrier include stabilizers such as human serum albumin (HSA), bulking agents such as carbohydrates, amino acids and polypeptides; pH adjusters or buffers; salts such as sodium chloride; and the like. These carriers may be in a crystalline or amorphous form or may be a mixture of the two.

Bulking agents that are particularly valuable include compatible carbohydrates, polypeptides, amino acids or combinations thereof. Suitable carbohydrates include monosaccharides such as galactose, D-mannose, sorbose, and the like; disaccharides, such as lactose, trehalose, and the like; cyclodextrins, such as 2-hydroxypropyl-.beta.-cyclodextrin;

and polysaccharides, such as raffinose, maltodextrins, dextrans, and the like; alditols, such as mannitol, xylitol, and the like. A preferred group of carbohydrates includes lactose, threhalose, raffinose maltodextrins, and mannitol. Suitable polypeptides include aspartame. Amino acids include alanine and glycine, with glycine being preferred.

Additives, which are minor components of the composition of this invention, may be included for conformational stability during spray drying and for improving dispersibility of the powder. These additives include hydrophobic amino acids such as tryptophan, tyrosine, leucine, phenylalanine, and the like.

Suitable pH adjusters or buffers include organic salts prepared from organic acids and bases, such as sodium citrate, sodium ascorbate, and the like; sodium citrate is preferred.

Pulmonary administration of a micellar iRNA formulation may be achieved through metered dose spray devices with propellants such as tetrafluoroethane, heptafluoroethane, dimethylfluoropropane, tetrafluoropropane, butane, isobutane, dimethyl ether and other non-CFC and CFC propellants.

Oral or Nasal Delivery

For ease of exposition the formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. Both the oral and nasal membranes offer advantages over other routes of administration. For example, drugs administered through these membranes have a rapid onset of action, provide therapeutic plasma levels, avoid first pass effect of hepatic metabolism, and avoid exposure of the drug to the hostile gastrointestinal (GI) environment. Additional advantages include easy access to the membrane sites so that the drug can be applied, localized and removed easily.

In oral delivery, compositions can be targeted to a surface of the oral cavity, e.g., to sublingual mucosa which includes the membrane of ventral surface of the tongue and the floor of the mouth or the buccal mucosa which constitutes the lining of the cheek. The sublingual mucosa is relatively permeable thus giving rapid absorption and acceptable bioavailability of many drugs. Further, the sublingual mucosa is convenient, acceptable and easily accessible.

The ability of molecules to permeate through the oral mucosa appears to be related to molecular size, lipid solubility and peptide protein ionization. Small molecules, less than 1000 daltons appear to cross mucosa rapidly. As molecular size increases, the permeability decreases rapidly. Lipid soluble compounds are more permeable than non-lipid soluble molecules. Maximum absorption occurs when molecules are un-ionized or neutral in electrical charges. Therefore charged molecules present the biggest challenges to absorption through the oral mucosae.

A pharmaceutical composition of iRNA may also be administered to the buccal cavity of a human being by spraying into the cavity, without inhalation, from a metered dose spray dispenser, a mixed micellar pharmaceutical formulation as described above and a propellant. In one embodiment, the dispenser is first shaken prior to spraying the pharmaceutical formulation and propellant into the buccal cavity.

Devices

For ease of exposition the devices, formulations, compositions and methods in this section are discussed largely with regard to unmodified iRNA agents. It should be understood, however, that these devices, formulations, compositions and methods can be practiced with other iRNA agents, e.g., modified iRNA agents, and such practice is within the invention. An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can be disposed on or in a device, e.g., a device which implanted or otherwise placed in a subject. Exemplary devices include devices which are introduced into the vasculature, e.g., devices inserted into the lumen of a vascular tissue, or which devices themselves form a part of the vasculature, including stents, catheters, heart valves, and other vascular devices. These devices, e.g., catheters or stents, can be placed in the vasculature of the lung, heart, or leg.

Other devices include non-vascular devices, e.g., devices implanted in the peritoneum, or in organ or glandular tissue, e.g., artificial organs. The device can release a therapeutic substance in addition to a iRNA, e.g., a device can release insulin.

Other devices include artificial joints, e.g., hip joints, and other orthopedic implants.

In one embodiment, unit doses or measured doses of a composition that includes iRNA are dispensed by an implanted device. The device can include a sensor that monitors a parameter within a subject. For example, the device can include pump, e.g., and, optionally, associated electronics.

Tissue, e.g., cells or organs can be treated with An iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) ex vivo and then administered or implanted in a subject.

The tissue can be autologous, allogeneic, or xenogeneic tissue. E.g., tissue can be treated to reduce graft v. host disease. In other embodiments, the tissue is allogeneic and the tissue is treated to treat a disorder characterized by unwanted gene expression in that tissue. E.g., tissue, e.g., hematopoietic cells, e.g., bone marrow hematopoietic cells, can be treated to inhibit unwanted cell proliferation.

Introduction of treated tissue, whether autologous or transplant, can be combined with other therapies.

In some implementations, the iRNA treated cells are insulated from other cells, e.g., by a semi-permeable porous barrier that prevents the cells from leaving the implant, but enables molecules from the body to reach the cells and molecules produced by the cells to enter the body. In one embodiment, the porous barrier is formed from alginate.

In one embodiment, a contraceptive device is coated with or contains an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). Exemplary devices include condoms, diaphragms, IUD (implantable uterine devices, sponges, vaginal sheaths, and birth control devices. In one embodiment, the iRNA is chosen to inactive sperm or egg. In another embodiment, the iRNA is chosen to be complementary to a viral or pathogen RNA, e.g., an RNA of an STD. In some instances, the iRNA composition can include a spermicide.

Dosage

In one aspect, the invention features a method of administering an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, to a subject (e.g., a human subject). The method includes administering a unit dose of the iRNA agent, e.g., a sRNA agent, e.g., double stranded sRNA agent that (a) the double-stranded part is 19-25 nucleotides (nt) long, preferably 21-23 nt, (b) is complementary to a target RNA (e.g., an endogenous or pathogen target RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotide long. In one embodiment, the unit dose is less than 1.4 mg per kg of bodyweight, or less than 10, 5, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, 0.00005 or 0.00001 mg per kg of bodyweight, and less than 200 nmole of RNA agent (e.g. about 4.4×10¹⁶ copies) per kg of bodyweight, or less than 1500, 750, 300, 150, 75, 15, 7.5, 1.5, 0.75, 0.15, 0.075, 0.015, 0.0075, 0.0015, 0.00075, 0.00015 nmole of RNA agent per kg of bodyweight.

The defined amount can be an amount effective to treat or prevent a disease or disorder, e.g., a disease or disorder associated with the target RNA. The unit dose, for example, can be administered by injection (e.g., intravenous or intramuscular), an inhaled dose, or a topical application. Particularly preferred dosages are less than 2, 1, or 0.1 mg/kg of body weight.

In a preferred embodiment, the unit dose is administered less frequently than once a day, e.g., less than every 2, 4, 8 or 30 days. In another embodiment, the unit dose is not administered with a frequency (e.g., not a regular frequency). For example, the unit dose may be administered a single time.

In one embodiment, the effective dose is administered with other traditional therapeutic modalities. In one embodiment, the subject has a viral infection and the modality is an antiviral agent other than an iRNA agent, e.g., other than a double-stranded iRNA agent, or sRNA agent,. In another embodiment, the subject has atherosclerosis and the effective dose of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, is administered in combination with, e.g., after surgical intervention, e.g., angioplasty.

In one embodiment, a subject is administered an initial dose and one or more maintenance doses of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). The maintenance dose or doses are generally lower than the initial dose, e.g., one-half less of the initial dose. A maintenance regimen can include treating the subject with a dose or doses ranging from 0.01 μg to 1.4 mg/kg of body weight per day, e.g., 10, 1, 0.1, 0.01, 0.001, or 0.00001 mg per kg of bodyweight per day. The maintenance doses are preferably administered no more than once every 5, 10, or 30 days. Further, the treatment regimen may last for a period of time which will vary depending upon the nature of the particular disease, its severity and the overall condition of the patient. In preferred embodiments the dosage may be delivered no more than once per day, e.g., no more than once per 24, 36, 48, or more hours, e.g., no more than once for every 5 or 8 days. Following treatment, the patient can be monitored for changes in his condition and for alleviation of the symptoms of the disease state. The dosage of the compound may either be increased in the event the patient does not respond significantly to current dosage levels, or the dose may be decreased if an alleviation of the symptoms of the disease state is observed, if the disease state has been ablated, or if undesired side-effects are observed.

The effective dose can be administered in a single dose or in two or more doses, as desired or considered appropriate under the specific circumstances. If desired to facilitate repeated or frequent infusions, implantation of a delivery device, e.g., a pump, semi-permanent stent (e.g., intravenous, intraperitoneal, intracisternal or intracapsular), or reservoir may be advisable.

In one embodiment, the iRNA agent pharmaceutical composition includes a plurality of iRNA agent species. In another embodiment, the iRNA agent species has sequences that are non-overlapping and non-adjacent to another species with respect to a naturally occurring target sequence. In another embodiment, the plurality of iRNA agent species is specific for different naturally occurring target genes. In another embodiment, the iRNA agent is allele specific.

In some cases, a patient is treated with a iRNA agent in conjunction with other therapeutic modalities. For example, a patient being treated for a viral disease, e.g. an HIV associated disease (e.g., AIDS), may be administered a iRNA agent specific for a target gene essential to the virus in conjunction with a known antiviral agent (e.g., a protease inhibitor or reverse transcriptase inhibitor). In another example, a patient being treated for cancer may be administered a iRNA agent specific for a target essential for tumor cell proliferation in conjunction with a chemotherapy.

Following successful treatment, it may be desirable to have the patient undergo maintenance therapy to prevent the recurrence of the disease state, wherein the compound of the invention is administered in maintenance doses, ranging from 0.01 μg to 100 g per kg of body weight (see U.S. Pat. No. 6,107,094).

The concentration of the iRNA agent composition is an amount sufficient to be effective in treating or preventing a disorder or to regulate a physiological condition in humans. The concentration or amount of iRNA agent administered will depend on the parameters determined for the agent and the method of administration, e.g. nasal, buccal, pulmonary. For example, nasal formulations tend to require much lower concentrations of some ingredients in order to avoid irritation or burning of the nasal passages. It is sometimes desirable to dilute an oral formulation up to 10-100 times in order to provide a suitable nasal formulation.

Certain factors may influence the dosage required to effectively treat a subject, including but not limited to the severity of the disease or disorder, previous treatments, the general health and/or age of the subject, and other diseases present. Moreover, treatment of a subject with a therapeutically effective amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) can include a single treatment or, preferably, can include a series of treatments. It will also be appreciated that the effective dosage of a iRNA agent such as a sRNA agent used for treatment may increase or decrease over the course of a particular treatment. Changes in dosage may result and become apparent from the results of diagnostic assays as described herein. For example, the subject can be monitored after administering a iRNA agent composition. Based on information from the monitoring, an additional amount of the iRNA agent composition can be administered.

Dosing is dependent on severity and responsiveness of the disease condition to be treated, with the course of treatment lasting from several days to several months, or until a cure is effected or a diminution of disease state is achieved. Optimal dosing schedules can be calculated from measurements of drug accumulation in the body of the patient. Persons of ordinary skill can easily determine optimum dosages, dosing methodologies and repetition rates. Optimum dosages may vary depending on the relative potency of individual compounds, and can generally be estimated based on EC50s found to be effective in in vitro and in vivo animal models. In some embodiments, the animal models include transgenic animals that express a human gene, e.g. a gene that produces a target RNA. The transgenic animal can be deficient for the corresponding endogenous RNA. In another embodiment, the composition for testing includes a iRNA agent that is complementary, at least in an internal region, to a sequence that is conserved between the target RNA in the animal model and the target RNA in a human.

The inventors have discovered that iRNA agents described herein can be administered to mammals, particularly large mammals such as nonhuman primates or humans in a number of ways.

In one embodiment, the administration of the iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, composition is parenteral, e.g. intravenous (e.g., as a bolus or as a diffusible infusion), intradermal, intraperitoneal, intramuscular, intrathecal, intraventricular, intracranial, subcutaneous, transmucosal, buccal, sublingual, endoscopic, rectal, oral, vaginal, topical, pulmonary, intranasal, urethral or ocular. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. Selected modes of delivery are discussed in more detail below.

The invention provides methods, compositions, and kits, for rectal administration or delivery of iRNA agents described herein.

Accordingly, an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent , or a DNA which encodes a an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) described herein, e.g., a therapeutically effective amount of a iRNA agent described herein, e.g., a iRNA agent having a double stranded region of less than 40, and preferably less than 30 nucleotides and having one or two 1-3 nucleotide single strand 3′ overhangs can be administered rectally, e.g., introduced through the rectum into the lower or upper colon. This approach is particularly useful in the treatment of, inflammatory disorders, disorders characterized by unwanted cell proliferation, e.g., polyps, or colon cancer.

The medication can be delivered to a site in the colon by introducing a dispensing device, e.g., a flexible, camera-guided device similar to that used for inspection of the colon or removal of polyps, which includes means for delivery of the medication.

The rectal administration of the iRNA agent is by means of an enema. The iRNA agent of the enema can be dissolved in a saline or buffered solution. The rectal administration can also by means of a suppository, which can include other ingredients, e.g., an excipient, e.g., cocoa butter or hydropropylmethylcellulose.

Any of the iRNA agents described herein can be administered orally, e.g., in the form of tablets, capsules, gel capsules, lozenges, troches or liquid syrups. Further, the composition can be applied topically to a surface of the oral cavity.

Any of the iRNA agents described herein can be administered buccally. For example, the medication can be sprayed into the buccal cavity or applied directly, e.g., in a liquid, solid, or gel form to a surface in the buccal cavity. This administration is particularly desirable for the treatment of inflammations of the buccal cavity, e.g., the gums or tongue, e.g., in one embodiment, the buccal administration is by spraying into the cavity, e.g., without inhalation, from a dispenser, e.g., a metered dose spray dispenser that dispenses the pharmaceutical composition and a propellant.

Any of the iRNA agents described herein can be administered to ocular tissue. For example, the medications can be applied to the surface of the eye or nearby tissue, e.g., the inside of the eyelid. They can be applied topically, e.g., by spraying, in drops, as an eyewash, or an ointment. Administration can be provided by the subject or by another person, e.g., a health care provider. The medication can be provided in measured doses or in a dispenser which delivers a metered dose. The medication can also be administered to the interior of the eye, and can be introduced by a needle or other delivery device which can introduce it to a selected area or structure. Ocular treatment is particularly desirable for treating inflammation of the eye or nearby tissue.

Any of the iRNA agents described herein can be administered directly to the skin. For example, the medication can be applied topically or delivered in a layer of the skin, e.g., by the use of a microneedle or a battery of microneedles which penetrate into the skin, but preferably not into the underlying muscle tissue. Administration of the iRNA agent composition can be topical. Topical applications can, for example, deliver the composition to the dermis or epidermis of a subject. Topical administration can be in the form of transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids or powders. A composition for topical administration can be formulated as a liposome, micelle, emulsion, or other lipophilic molecular assembly. The transdermal administration can be applied with at least one penetration enhancer, such as iontophoresis, phonophoresis, and sonophoresis.

Any of the iRNA agents described herein can be administered to the pulmonary system. Pulmonary administration can be achieved by inhalation or by the introduction of a delivery device into the pulmonary system, e.g., by introducing a delivery device which can dispense the medication. A preferred method of pulmonary delivery is by inhalation. The medication can be provided in a dispenser which delivers the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Pulmonary delivery is effective not only for disorders which directly affect pulmonary tissue, but also for disorders which affect other tissue.

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or aerosol for pulmonary delivery.

Any of the iRNA agents described herein can be administered nasally. Nasal administration can be achieved by introduction of a delivery device into the nose, e.g., by introducing a delivery device which can dispense the medication. Methods of nasal delivery include spray, aerosol, liquid, e.g., by drops, or by topical administration to a surface of the nasal cavity. The medication can be provided in a dispenser with delivery of the medication, e.g., wet or dry, in a form sufficiently small such that it can be inhaled. The device can deliver a metered dose of medication. The subject, or another person, can administer the medication.

Nasal delivery is effective not only for disorders which directly affect nasal tissue, but also for disorders which affect other tissue

iRNA agents can be formulated as a liquid or nonliquid, e.g., a powder, crystal, or for nasal delivery.

An iRNA agent can be packaged in a viral natural capsid or in a chemically or enzymatically produced artificial capsid or structure derived therefrom.

The dosage of a pharmaceutical composition including a iRNA agent can be administered in order to alleviate the symptoms of a disease state, e.g., cancer or a cardiovascular disease. A subject can be treated with the pharmaceutical composition by any of the methods mentioned above.

Gene expression in a subject can be modulated by administering a pharmaceutical composition including an iRNA agent.

A subject can be treated by administering a defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent) composition that is in a powdered form, e.g., a collection of microparticles, such as crystalline particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

A subject can be treated by administering a defined amount of an iRNA agent composition that is prepared by a method that includes spray-drying, i.e. atomizing a liquid solution, emulsion, or suspension, immediately exposing the droplets to a drying gas, and collecting the resulting porous powder particles. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

The iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), can be provided in a powdered, crystallized or other finely divided form, with or without a carrier, e.g., a micro- or nano-particle suitable for inhalation or other pulmonary delivery. This can include providing an aerosol preparation, e.g., an aerosolized spray-dried composition. The aerosol composition can be provided in and/or dispensed by a metered dose delivery device.

The subject can be treated for a condition treatable by inhalation, e.g., by aerosolizing a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition and inhaling the aerosolized composition. The iRNA agent can be an sRNA. The composition can include a plurality of iRNA agents, e.g., specific for one or more different endogenous target RNAs. The method can include other features described herein.

A subject can be treated by, for example, administering a composition including an effective/defined amount of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), wherein the composition is prepared by a method that includes spray-drying, lyophilization, vacuum drying, evaporation, fluid bed drying, or a combination of these techniques.

In another aspect, the invention features a method that includes: evaluating a parameter related to the abundance of a transcript in a cell of a subject; comparing the evaluated parameter to a reference value; and if the evaluated parameter has a preselected relationship to the reference value (e.g., it is greater), administering a iRNA agent (or a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes a iRNA agent or precursor thereof) to the subject. In one embodiment, the iRNA agent includes a sequence that is complementary to the evaluated transcript. For example, the parameter can be a direct measure of transcript levels, a measure of a protein level, a disease or disorder symptom or characterization (e.g., rate of cell proliferation and/or tumor mass, viral load,)

In another aspect, the invention features a method that includes: administering a first amount of a composition that comprises an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a subject, wherein the iRNA agent includes a strand substantially complementary to a target nucleic acid; evaluating an activity associated with a protein encoded by the target nucleic acid; wherein the evaluation is used to determine if a second amount should be administered. In a preferred embodiment the method includes administering a second amount of the composition, wherein the timing of administration or dosage of the second amount is a function of the evaluating. The method can include other features described herein.

In another aspect, the invention features a method of administering a source of a double-stranded iRNA agent (ds iRNA agent) to a subject. The method includes administering or implanting a source of a ds iRNA agent, e.g., a sRNA agent, that (a) includes a double-stranded region that is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to a target RNA (e.g., an endogenous RNA or a pathogen RNA), and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the source releases ds iRNA agent over time, e.g. the source is a controlled or a slow release source, e.g., a microparticle that gradually releases the ds iRNA agent. In another embodiment, the source is a pump, e.g., a pump that includes a sensor or a pump that can release one or more unit doses.

In one aspect, the invention features a pharmaceutical composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) including a nucleotide sequence complementary to a target RNA, e.g., substantially and/or exactly complementary. The target RNA can be a transcript of an endogenous human gene. In one embodiment, the iRNA agent (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nt long. In one embodiment, the pharmaceutical composition can be an emulsion, microemulsion, cream, jelly, or liposome.

In one example the pharmaceutical composition includes an iRNA agent mixed with a topical delivery agent. The topical delivery agent can be a plurality of microscopic vesicles. The microscopic vesicles can be liposomes. In a preferred embodiment the liposomes are cationic liposomes.

In another aspect, the pharmaceutical composition includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) admixed with a topical penetration enhancer. In one embodiment, the topical penetration enhancer is a fatty acid. The fatty acid can be arachidonic acid, oleic acid, lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid, stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate, monolein, dilaurin, glyceryl 1-monocaprate, 1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or a C₁₋₁₀ alkyl ester, monoglyceride, diglyceride or pharmaceutically acceptable salt thereof.

In another embodiment, the topical penetration enhancer is a bile salt. The bile salt can be cholic acid, dehydrocholic acid, deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid, taurocholic acid, taurodeoxycholic acid, chenodeoxycholic acid, ursodeoxycholic acid, sodium tauro-24,25-dihydro-fusidate, sodium glycodihydrofusidate, polyoxyethylene-9-lauryl ether or a pharmaceutically acceptable salt thereof

In another embodiment, the penetration enhancer is a chelating agent. The chelating agent can be EDTA, citric acid, a salicyclate, a N-acyl derivative of collagen, laureth-9, an N-amino acyl derivative of a beta-diketone or a mixture thereof.

In another embodiment, the penetration enhancer is a surfactant, e.g., an ionic or nonionic surfactant. The surfactant can be sodium lauryl sulfate, polyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether, a perfluorchemical emulsion or mixture thereof.

In another embodiment, the penetration enhancer can be selected from a group consisting of unsaturated cyclic ureas, 1-alkyl-alkones, 1-alkenylazacyclo-alakanones, steroidal anti-inflammatory agents and mixtures thereof In yet another embodiment the penetration enhancer can be a glycol, a pyrrol, an azone, or a terpenes.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a form suitable for oral delivery. In one embodiment, oral delivery can be used to deliver an iRNA agent composition to a cell or a region of the gastro-intestinal tract, e.g., small intestine, colon (e.g., to treat a colon cancer), and so forth. The oral delivery form can be tablets, capsules or gel capsules. In one embodiment, the iRNA agent of the pharmaceutical composition modulates expression of a cellular adhesion protein, modulates a rate of cellular proliferation, or has biological activity against eukaryotic pathogens or retroviruses. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methylcellulose phthalate or cellulose acetate phthalate.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer. The penetration enhancer can be a bile salt or a fatty acid. The bile salt can be ursodeoxycholic acid, chenodeoxycholic acid, and salts thereof. The fatty acid can be capric acid, lauric acid, and salts thereof

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent and a delivery vehicle. In one embodiment, the iRNA agent is (a) is 19-25 nucleotides long, preferably 21-23 nucleotides, (b) is complementary to an endogenous target RNA, and, optionally, (c) includes at least one 3′ overhang 1-5 nucleotides long.

In one embodiment, the delivery vehicle can deliver an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) to a cell by a topical route of administration. The delivery vehicle can be microscopic vesicles. In one example the microscopic vesicles are liposomes. In a preferred embodiment the liposomes are cationic liposomes. In another example the microscopic vesicles are micelles.In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in an injectable dosage form. In one embodiment, the injectable dosage form of the pharmaceutical composition includes sterile aqueous solutions or dispersions and sterile powders. In a preferred embodiment the sterile solution can include a diluent such as water; saline solution; fixed oils, polyethylene glycols, glycerin, or propylene glycol.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in oral dosage form. In one embodiment, the oral dosage form is selected from the group consisting of tablets, capsules and gel capsules. In another embodiment, the pharmaceutical composition includes an enteric material that substantially prevents dissolution of the tablets, capsules or gel capsules in a mammalian stomach. In a preferred embodiment the enteric material is a coating. The coating can be acetate phthalate, propylene glycol, sorbitan monoleate, cellulose acetate trimellitate, hydroxy propyl methyl cellulose phthalate or cellulose acetate phthalate. In one embodiment, the oral dosage form of the pharmaceutical composition includes a penetration enhancer, e.g., a penetration enhancer described herein.

In another embodiment, the oral dosage form of the pharmaceutical composition includes an excipient. In one example the excipient is polyethyleneglycol. In another example the excipient is precirol.

In another embodiment, the oral dosage form of the pharmaceutical composition includes a plasticizer. The plasticizer can be diethyl phthalate, triacetin dibutyl sebacate, dibutyl phthalate or triethyl citrate.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a rectal dosage form. In one embodiment, the rectal dosage form is an enema. In another embodiment, the rectal dosage form is a suppository.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a vaginal dosage form. In one embodiment, the vaginal dosage form is a suppository. In another embodiment, the vaginal dosage form is a foam, cream, or gel.

In one aspect, the invention features a pharmaceutical composition including an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) in a pulmonary or nasal dosage form. In one embodiment, the iRNA agent is incorporated into a particle, e.g., a macroparticle, e.g., a microsphere. The particle can be produced by spray drying, lyophilization, evaporation, fluid bed drying, vacuum drying, or a combination thereof. The microsphere can be formulated as a suspension, a powder, or an implantable solid.

In one aspect, the invention features a spray-dried iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof) composition suitable for inhalation by a subject, including: (a) a therapeutically effective amount of a iRNA agent suitable for treating a condition in the subject by inhalation; (b) a pharmaceutically acceptable excipient selected from the group consisting of carbohydrates and amino acids; and (c) optionally, a dispersibility-enhancing amount of a physiologically-acceptable, water-soluble polypeptide.

In one embodiment, the excipient is a carbohydrate. The carbohydrate can be selected from the group consisting of monosaccharides, disaccharides, trisaccharides, and polysaccharides. In a preferred embodiment the carbohydrate is a monosaccharide selected from the group consisting of dextrose, galactose, mannitol, D-mannose, sorbitol, and sorbose. In another preferred embodiment the carbohydrate is a disaccharide selected from the group consisting of lactose, maltose, sucrose, and trehalose.

In another embodiment, the excipient is an amino acid. In one embodiment, the amino acid is a hydrophobic amino acid. In a preferred embodiment the hydrophobic amino acid is selected from the group consisting of alanine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, and valine. In yet another embodiment the amino acid is a polar amino acid. In a preferred embodiment the amino acid is selected from the group consisting of arginine, histidine, lysine, cysteine, glycine, glutamine, serine, threonine, tyrosine, aspartic acid and glutamic acid.

In one embodiment, the dispersibility-enhancing polypeptide is selected from the group consisting of human serum albumin, a-lactalbumin, trypsinogen, and polyalanine.

In one embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter (MMD) of less than 10 microns. In another embodiment, the spray-dried iRNA agent composition includes particles having a mass median diameter of less than 5 microns. In yet another embodiment the spray-dried iRNA agent composition includes particles having a mass median aerodynamic diameter (MMAD) of less than 5 microns.

In certain other aspects, the invention provides kits that include a suitable container containing a pharmaceutical formulation of an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof). In certain embodiments the individual components of the pharmaceutical formulation may be provided in one container. Alternatively, it may be desirable to provide the components of the pharmaceutical formulation separately in two or more containers, e.g., one container for an iRNA agent preparation, and at least another for a carrier compound. The kit may be packaged in a number of different configurations such as one or more containers in a single box. The different components can be combined, e.g., according to instructions provided with the kit. The components can be combined according to a method described herein, e.g., to prepare and administer a pharmaceutical composition. The kit can also include a delivery device.

In another aspect, the invention features a device, e.g., an implantable device, wherein the device can dispense or administer a composition that includes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, (e.g., a precursor, e.g., a larger iRNA agent which can be processed into a sRNA agent, or a DNA which encodes an iRNA agent, e.g., a double-stranded iRNA agent, or sRNA agent, or precursor thereof), e.g., a iRNA agent that silences an endogenous transcript. In one embodiment, the device is coated with the composition. In another embodiment the iRNA agent is disposed within the device. In another embodiment, the device includes a mechanism to dispense a unit dose of the composition. In other embodiments the device releases the composition continuously, e.g., by diffusion. Exemplary devices include stents, catheters, pumps, artificial organs or organ components (e.g., artificial heart, a heart valve, etc.), and sutures.

As used herein, the term “crystalline” describes a solid having the structure or characteristics of a crystal, i.e., particles of three-dimensional structure in which the plane faces intersect at definite angles and in which there is a regular internal structure. The compositions of the invention may have different crystalline forms. Crystalline forms can be prepared by a variety of methods, including, for example, spray drying.

The invention is further illustrated by the following examples, which should not be construed as further limiting.

EXAMPLES Example 1 Inhibition of Endogenous ApoM Gene Expression in Mice

Apolipoprotein M (ApoM) is a human apolipoprotein predominantly present in high-density lipoprotein (HDL) in plasma. ApoM is reported to be expressed exclusively in liver and in kidney (Xu N et al., Biochem J Biol Chem 1999 Oct. 29; 274(44):31286-90). Mouse ApoM is a 21kD membrane associated protein, and, in serum, the protein is associated with HDL particles. ApoM gene expression is regulated by the transcription factor hepatocyte nuclear factor 1 alpha (Hnf-la), as Hnf-1α^(−/−) mice are ApoM deficient. In humans, mutations in the HNF-1 alpha gene represent a common cause of maturity-onset diabetes of the young (MODY).

A variety of test iRNAs were synthesized to target the mouse ApoM gene. This gene was chosen in part because of its high expression levels and exclusive activity in the liver and kidney.

Three different classes of dsRNA agents were synthesized, each class having different modifications and features at the 5′ and 3′ ends, see Table 4.

TABLE 4 Targeted ORF's  5 The23mer: AAGTTTGGGCAGCTCTGCTCT (SEQ ID NO: 6708) 19 The23mer: AAGTGGACATACCGATTGACT (SEQ ID NO: 6709) 25 The23mer: AACTCAGAACTGAAGGGCGCC (SEQ ID NO: 6710) 27 The23mer: AAGGGCGCCCAGACATGAAAA (SEQ ID NO: 6711) 3'-UTR (beginning at 645) 42: AAGATAGGAGCCCAGCTTCGA (SEQ ID NO: 6712) Class I 21-nt iRNAs, t, deoxythymidine; p, phosphate pGUUUGGGCAGCUCUGCUCUtt (SEQ ID NO: 6712) #1 pAGAGCAGAGCUGCCCAAACtt (SEQ ID NO: 6713) pGUGGACAUACCGAUUGACUtt (SEQ ID NO: 6714) #2 pAGUCAAUCGGUAUGUCCACtt (SEQ ID NO: 6715) pCUCAGAACUGAAGGGCGCCtt (SEQ ID NO: 6716) #3 pGGCGCCCUUCAGUUCUGAGtt (SEQ ID NO: 6717) pGAUAGGAGCCCAGCUUCGAtt (SEQ ID NO: 6718) #4 pUCGAAGCUGGGCUCCUAUCtt (SEQ ID NO: 6719) Class II 21-nt iRNAs, t, deoxythymidine; p, phosphate; ps, thiophosphate pGUUUGGGCAGCUCUGCUCpsUpstpst (SEQ ID NO: 6720) #11 pAGAGCAGAGCUGCCCAAApsCpstpst (SEQ ID NO: 6721) pGUGGACAUACCGAUUGACpsUpstpst (SEQ ID NO: 6722) #13 pAGUCAAUCGGUAUGUCCApsCpstpst (SEQ ID NO: 6723) pCUCAGAACUGAAGGGCGCpsCpstpst (SEQ ID NO: 6724) #15 pGGCGCCCUUCAGUUCUGApsGpstpst (SEQ ID NO: 6725) pGAUAGGAGCCCAGCUUCGpsApstpst (SEQ ID NO: 6726) #17 pUCGAAGCUGGGCUCCUAUpsCpstpst (SEQ ID NO: 6727) Class III 23-nt antisense, 21-nt sense, blunt-ended 5′-as GUUUGGGCAGCUCUGCUCUCU (SEQ ID NO: 6728) #19 AGAGAGCAGAGCUGCCCAAACUU (SEQ ID NO: 6729) GUGGACAUACCGAUUGACUGA (SEQ ID NO: 6730) #21 UCAGUCAAUCGGUAUGUCCACUU (SEQ ID NO: 6731) CUCAGAACUGAAGGGCGCCCA (SEQ ID NO: 6732) #23 PUGGGCGCCCUUCAGUUCUGAGUU (SEQ ID NO: 6733) GAUAGGAGCCCAGCUUCGAGU (SEQ ID NO: 6734) #25 ACUCGAAGCUGGGCUCCUAUCUU (SEQ ID NO: 6735)

Class I dsRNAs consisted of 21 nucleotide paired sense and antisense strands. The sense and antisense strands were each phosphorylated at their 5′ ends. The double stranded region was 19 nucleotides long and consisted of ribonucleotides. The 3′ end of each strand created a two nucleotide overhang consisting of two deoxyribonucleotide thymidines. See constructs #1-4 in Table 4.

Class II dsRNAs were also 21 nucleotides long, with a 19 nucleotide double strand region. The sense and antisense strands were each phosphorylated at their 5′ ends. The three 3′ terminal nucleotides of the sense and antisense strands were phosphorothioate deoxyribonucleotides, and the two terminal phosphorothioate thymidines were unpaired, creating a 3′ overhang region at each end of the iRNA molecule. See constructs 11, 13, 15, and 17 in Table 4.

Class III dsRNAs included a 23 ribonucleotide antisense strand and a 21 ribonucleotide sense strand, to form a construct having a blunt 5′ and a 3′ overhang region. See constructs 19, 21, 23, and 25 in Table 4.

Within each of the three classes of iRNAs, the four dsRNA molecules were designed to target four different regions of the ApoM transcript. dsRNAs 1, 11, and 19 targeted the 5′ end of the open reading frame (ORF). dsRNAs 2, 13, and 21, and 3, 15, and 23, targeted two internal regions (one 5′ proximal and one 3′ proximal) of the ORF, and the 4, 17, and 25 iRNA constructs targeted to a region of the 3′ untranslated sequence (3′ UTS) of the ApoM mRNA. This is summarized in Table 5.

TABLE 5 iRNA molecules targeted to mouse ApoM iRNA targeted iRNA targeted iRNA targeted to 5′ end to middle ORF to middle ORF iRNA targeted of ORF (5′ proximal) (3′ proximal) to 3′UTS Class 1 2 3 4 I Class 11 13 15 17 II Class 19 21 23 25 III

CD1 mice (6-8 weeks old, ˜35 g) were administered one of the test iRNAs in PBS solution. Two hundred micrograms of iRNA in a volume of solution equal to 10% body weight (˜5.7 mg iRNA/kg mouse) was administered by the method of high pressure tail vein injection, over a 10-20 sec. time interval. After a 24 h recovery period, a second injection was performed using the same dose and mode of administration as the first injection, and following another 24 h, a third and final injection was administered, also using the same dose and mode of administration. After a final 24 h recovery, the mouse was sacrificed, serum was collected and the liver and kidney harvested to assay for an affect on ApoM gene expression. Expression was monitored by quantitative RT-PCR and Western blot analyses. This experiment was repeated for each of the iRNAs listed in table 4.

Class I iRNAs did not alter ApoM RNA levels in mice, as indicated by quantitative RT-PCR. This is in contrast to the effect of these iRNAs in cultured HepG2 cells. Cells cotransfected with a plasmid expressing exogenous ApoM RNA under a CMV promoter and a class I iRNA demonstrated a 25% or greater reduction in ApoM RNA concentrations as compared to control transfections. The iRNA molecules 1, 2 and 3 each caused a 75% decrease in exogenous ApoM mRNA levels.

Class II iRNAs reduced liver and kidney ApoM mRNA levels by ˜30-85%. The iRNA molecule “13” elicited the most dramatic reduction in mRNA levels; quantitative RT-PCR indicated a decrease of about 85% in liver tissue. Serum ApoM protein levels were also reduced as was evidenced by Western blot analysis. The iRNAs 11, 13 and 15, reduced protein levels by about 50%, while iRNA 17 had the mildest effect, reducing levels only by ˜15-20%.

Class III iRNAs (constructs 19, 21, and 23) reduced serum Apo levels by ˜40-50%.

To determine the effect of dosage on iRNA mediated ApoM inhibition, the experiment described above was repeated with three injections of 50 μg iRNA “11” (˜1.4mg iRNA/kg mouse). This lower dosage of iRNA resulted in a reduction of serum ApoM levels of about 50%. This is compared with the reduction seen with the 200 μg injections, which reduced serum levels by 25-45%. These results indicated the lower dosage amounts of iRNAs were effective.

In an effort to increase iRNA uptake by cells, iRNAs were precomplexed with lipofectamine prior to tail vein injections. ApoM protein levels were about 50% of wildtype levels in mice injected with iRNA “11” when the molecules were preincubated with lipofectamine; ApoM levels were also about 50% of wildtype when mice were injected with iRNA “11” that was not precomplexed with lipofectamine.

These experiments revealed that modified iRNAs can greatly influence RNAi-mediated gene silencing. As demonstrated herein, modifications including phosphorothioate nucleotides are particularly effective at decreasing target protein levels.

Example 2 apoB Protein as a Therapeutic Target for Lipid-based Diseases

Apolipoprotein B (apoB) is a candidate target gene for the development of novel therapies for lipid-based diseases.

Methods described herein can be used to evaluate the efficacy of a particular siRNA as a therapeutic tool for treating lipid metabolism disorders resulting elevated apoB levels. Use of siRNA duplexes to selectively bind and inactivate the target apoB mRNA is an approach totreat these disorders.

Two approaches:

i) Inhibition of apoB in ex-vivo models by transfecting siRNA duplexes homologous to human apoB mRNA in a human hepatoma cell line (Hep G2) and monitor the level of the protein and the RNA using the Western blotting and RT-PCR methods, respectively. siRNA molecules that efficiently inhibit apoB expression will be tested for similar effects in vivo.

ii) In vivo trials using an apoB transgenic mouse model (apoB100 Transgenic Mice, C57BL/6NTac-TgN (APOB100), Order Model #'s:1004-T (hemizygotes), B6 (control)). siRNA duplexes are designed to target apoB-100 or CETP/apoB double transgenic mice which express both cholesteryl ester transfer protein (CETP) and apoB. The effect of the siRNA on gene expression in vivo can be measured by monitoring the HDL/LDL cholesterol level in serum. The results of these experiments would indicate the therapeutic potential of siRNAs to treat lipid-based diseases, including hypercholesterolemia, HDL/LDL cholesterol imbalance, familial combined hyperlipidemia, and acquired hyperlipidemia.

Background Fats, in the form of triglycerides, are ideal for energy storage because they are highly reduced and anhydrous. An adipocyte (or fat cell) consists of a nucleus, a cell membrane, and triglycerides, and its function is to store triglycerides.

The lipid portion of the human diet consists largely of triglycerides and cholesterol (and its esters). These must be emulsified and digested to be absorbed. Specifically, fats (triacylglycerols) are ingested. Bile (bile acids, salts, and cholesterol), which is made in the liver, is secreted by the gall bladder. Pancreatic lipase digests the triglycerides to fatty acids, and also digests di-, and mono-acylglycerols, which are absorbed by intestinal epithelial cells and then are resynthesized into triacylglycerols once inside the cells. These triglycerides and some cholesterols are combined with apolipoproteins to produce chylomicrons. Chylomicrons consist of approximately 95% triglycerides. The chylomicrons transport fatty acids to peripheral tissues. Any excess fat is stored in adipose tissue.

Lipid transport and clearance from the blood into cells, and from the cells into the blood and the liver, is mediated by the lipoprotein transport proteins. This class of approximately 17 proteins can be divided into three groups: Apolipoproteins, lipoprotein processing proteins, and lipoprotein receptors.

Apolipoproteins coat lipoprotein particles, and include the A-I, A-II, A-IV, B, CI, CII, CIII, D, E, Apo(a) proteins. Lipoprotein processing proteins include lipoprotein lipase, hepatic lipase, lecithin cholesterol acyltransferase and cholesterol ester transfer protein. Lipoprotein receptors include the low density lipoprotein (LDL) receptor, chylomicron-remnant receptor (the LDL receptor like protein or LDL receptor related protein - LRP) and the scavenger receptor.

Lipoprotein Metabolism Since the triglycerides, cholesterol esters, and cholesterol absorbed into the small intestine are not soluble in aqueous medium, they must be combined with suitable proteins (apolipoproteins) in order to prevent them from forming large oil droplets. The resulting lipoproteins undergo a type of metabolism as they pass through the bloodstream and certain organs (notably the liver).

Also synthesized in the liver is high density lipoprotein (HDL), which contains the apoproteins A-1, A-2, C-1, and D; HDL collects cholesterol from peripheral tissues and blood vessels and returns it to the liver. LDL is taken up by specific cell surface receptors into an endosome, which fuses with a lysosome where cholesterol ester is converted to free cholesterol. The apoproteins (including apo B-100) are digested to amino acids. The receptor protein is recycled to the cell membrane.

The free cholesterol formed by this process has two fates. First, it can move to the endoplasmic reticulum (ER), where it can inhibit HMG-CoA reductase, the synthesis of HMG-CoA reductase, and the synthesis of cell surface receptors for LDL. Also in the ER, cholesterol can speed up the degradation of HMG-CoA reductase. The free cholesterol can also be converted by acyl-CoA and acyl transferase (ACAT) to cholesterol esters, which form oil droplets.

ApoB is the major apolipoprotein of chylomicrons of very low density lipoproteins (VLDL, which carry most of the plasma triglyceride) and low density lipoprotein (LDL, which carry most of the plasma cholesterol). ApoB exists in human plasma in two isoforms, apoB-48 and apoB-100.

ApoB-100 is the major physiological ligand for the LDL receptor. The ApoB precursor has 4563 amino acids, and the mature apoB-100 has 4536 amino acid residues. The LDL-binding domain of ApoB-100 is proposed to be located between residues 3129 and 3532. ApoB-100 is synthesized in the liver and is required for the assembly of very low density lipoproteins VLDL and for the preparation of apoB-100 to transport triglycerides (TG) and cholesterol from the liver to other tissues. ApoB-100 does not interchange between lipoprotein particles, as do the other lipoproteins, and it is found in IDL and LDL particles. After the removal of apolipoproteins A, E and C, apoB is incorporation into VLDL by hepatocytes. ApoB-48 is present in chylomicrons and plays an essential role in the intestinal absorption of dietary fats. ApoB-48 is synthesized in the small intestine. It comprises the N-terminal 48% of apoB-100 and is produced by a posttranscriptional apoB-100 mRNA editing event at codon 2153 (C to U). This editing event is a product of the apoBEC-lb enzyme, which is expressed in the intestine. This editing event creates a stop codon instead of a glutamine codon, and therefore apoB-48, instead of apoB-100 is expressed in the intestine (apoB-100 is expressed in the liver).

There is also strong evidence that plasma apoB levels may be a better index of the risk of coronary artery disease (CAD) than total or LDL cholesterol levels. Clinical studies have demonstrated the value of measuring apoB in hypertriglyceridemic, hypercholesterolemic and normalipidemic subjects.

TABLE 6 Reference Range Lipid level in the Blood Lipid Range (mmols/L) Plasma Cholesterol 3.5-6.5 Low density lipoprotein 1.55-4.4  Very low density lipoprotein 0.128-0.645 High density lipoprotein/triglycerides 0.5-2.1 Total lipid   4.0-10 g/L

Molecular genetics of lipid metabolism in both humans and induced mutant mouse models Elevated plasma levels of LDL and apoB are associated with a higher risk for atherosclerosis and coronary heart disease, a leading cause of mortality. ApoB is the mandatory constituent of LDL particles. In addition to its role in lipoprotein metabolism, apoB has also been implicated as a factor in male infertility and fetal development. Furthermore, two quantitative trait loci regulating plasma apoB levels have been discovered, through the use of transgenic mouse models. Future experiments will facilitate the identification of human orthologous genes encoding regulators of plasma apoB levels. These loci are candidate therapeutic targets for human disorders characterized by altered plasma apoB levels. Such disorders include non-apoB linked hypobetalipoproteinemia and familial combined hyperlipidemia. The identification of these genetic loci would also reveal possible new pathways involved in the regulation of apoB secretion, potentially providing novel sites for pharmacological therapy.

Diseases and Clinical Pharmacology Familial combined hyperlipemia (FCHL) affects an estimated one in 10 Americans. FCHL can cause premature heart disease.

Familial Hypercholesterolemia (high level of apo B) A common genetic disorder of lipid metabolism. Familial hypercholesterolemia is characterized by elevated serum TC in association with xanthelasma, tendon and tuberous xanthomas, accelerated atherosclerosis, and early death from myocardial infarction (MI). It is caused by absent or defective LDL cell receptors, resulting in delayed LDL clearance, an increase in plasma LDL levels, and an accumulation of LDL cholesterol in macrophages over joints and pressure points, and in blood vessels.

Atherosclerosis (high level of apo B) Atherosclerosis develops as a deposition of cholesterol and fat in the arterial wall due to disturbances in lipid transport and clearance from the blood into cells and from the cells to blood and the liver.

Clinical studies have demonstrated that elevation of total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C) and apoB-100 promote human atherosclerosis. Similarly, decreased levels of high-density lipoprotein cholesterol (HDL-C) are associated with the development of atherosclerosis.

ApoB may be factor in the genetic cause of high cholesterol.

The risk of coronary artery disease (CAD) (high level of apo B) Cardiovascular disease, including coronary heart disease and stroke, is a leading cause of death and disability. The major risk factors include age, gender, elevated low-density lipoprotein cholesterol blood levels, decreased high-density lipoprotein cholesterol levels, cigarette smoking, hypertension, and diabetes. Emerging risk factors include elevated lipoprotein (a), remnant lipoproteins, and C reactive protein. Dietary intake, physical activity and genetics also impact cardiovascular risk. Hypertension and age are the major risk factors for stroke.

Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of apoB-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP).

Model for human atherosclerosis (Lipoprotein A transgenic mouse) Numerous studies have demonstrated that an elevated plasma level of lipoprotein(a) (Lp(a)) is a major independent risk factor for coronary heart disease (CHD). Current therapies, however, have little or no effect on apo(a) levels and the homology between apo(a) and plasminogen presents barriers to drug development. Lp(a) particles consist of apo(a) and apoB-100 proteins, and they are found only in primates and the hedgehog. The development of LPA transgenic mouse requires the creation of animals that express both human apoB and apo(a) transgenes to achieve assembly of LP(a). An atherosclerosis mouse model would facilitate the study of the disease process and factors influencing it, and further would facilitate the development of therapeutic or preventive agents. There are several strategies for gene-oriented therapy. For example, the missing or non-functional gene can be replaced, or unwanted gene activity can be inhibited.

Model for lipid Metabolism and Atherosclerosis DNX Transgenic Sciences has demonstrated that both CETP/ApoB and ApoB transgenic mice develop atherosclerotic plaques.

Model for apoB-100 overexpression The apoB-100 transgenic mice express high levels of human apoB-100. They consequently demonstrate elevated serum levels of LDL cholesterol.

After 6 months on a high-fat diet, the mice develop significant foam cell accumulation under the endothelium and within the media, as well as cholesterol crystals and fibrotic lesions.

Model for Cholesteryl ester transfer protein over expression The apoB-100 transgenic mice express the human enzyme, CETP, and consequently demonstrate a dramatically reduced level of serum HDL cholesterol.

Model for apoB-100 and CETP overexpression The apoB-100 transgenic mice express both CETP and apoB-100, resulting in mice with a human like serum HDL/LDL distribution. Following 6 months on a high-fat diet these mice develop significant foam cell accumulation underlying the endothelium and within the media, as well as cholesterol crystals and fibrotic lesions.

ApoB100 Transgenic Mice (Order Model #'s: 1004-T (hemizygotes), B6 (control)) These mice express high levels of human apoB-100, resulting in mice with elevated serum levels of LDL cholesterol. These mice are useful in identifying and evaluating compounds to reduce elevated levels of LDL cholesterol and the risk of atherosclerosis. When fed a high fat cholesterol diet, these mice develop significant foam cell accumulation underly the endothelium and within the media, and have significantly more complex atherosclerotic lesions than control animals.

Double Transgenic Mice, CETP/ApoB100 (Order Model #: 1007-TT) These mice express both CETP and apoB-100, resulting in a human-like serum HDL/LDL distribution. These mice are useful for evaluating compounds to treat hypercholesterolemia or HDL/LDL cholesterol imbalance to reduce the risk of developing atherosclerosis. When fed a high fat high cholesterol diet, these mice develop significant foam cell accumulation underlying the endothelium and within the media, and have significantly more complex atherosclerotic lesions than control animals.

ApoE gene knockout mouse Homozygous apoE knockout mice exhibit strong hypercholesterolemia, primarily due to elevated levels of VLDL and IDL caused by a defect in lipoprotein clearance from plasma. These mice develop atherosclerotic lesions which progress with age and resemble human lesions (Zhang et al., Science 258:46-71, 1992; Plump etal., Cell 71:343-353, 1992; Nakashima et al., Arterioscler Thromp. 14:133-140, 1994; Reddick et al., Arterioscler Tromb. 14:141-147, 1994). These mice are a promising model for studying the effect of diet and drugs on atherosclerosis.

Low density lipoprotein receptor (LDLR) mediates lipoprotein clearance from plasma through the recognition of apoB and apoE on the surface of lipoprotein particles. Humans, who lack or have a decreased number of the LDL receptors, have familial hypercholesterolemia and develop CHD at an early age.

ApoE Knockout Mice (Order Model #: APOE-M) The apoE knockout mouse was created by gene targeting in embryonic stem cells to disrupt the apoE gene. ApoE, a glycoprotein, is a structural component of very low density lipoprotein (VLDL) synthesized by the liver and intestinally synthesized chylomicrons. It is also a constituent of a subclass of high density lipoproteins (HDLs) involved in cholesterol transport activity among cells. One of the most important roles of apoE is to mediate high affinity binding of chylomicrons and VLDL particles that contain apoE to the low density lipoprotein (LDL) receptor. This allows for the specific uptake of these particles by the liver which is necessary for transport preventing the accumulation in plasma of cholesterol-rich remnants. The homozygous inactivation of the apoE gene results in animals that are devoid of apoE in their sera. The mice appear to develop normally, but they exhibit five times the normal serum plasma cholesterol and spontaneous atherosclerotic lesions. This is similar to a disease in people who have a variant form of the apoE gene that is defective in binding to the LDL receptor and are at risk for early development of atherosclerosis and increased plasma triglyceride and cholesterol levels. There are indications that apoE is also involved in immune system regulation, nerve regeneration and muscle differentiation. The apoE knockout mice can be used to study the role of apoE in lipid metabolism, atherogenesis, and nerve injury, and to investigate intervention therapies that modify the atherogenic process.

Apoe4 Targeted Replacement Mouse (Order Model #: 001549-M) ApoE is a plasma protein involved in cholesterol transport, and the three human isoforms (E2, E3, and E4) have been associated with atherosclerosis and Alzheimer's disease. Gene targeting of 129 ES cells was used to replace the coding sequence of mouse apoE with human APOE4 without disturbing the murine regulatory sequences. The E4 isoform occurs in approximately 14% of the human population and is associated with increased plasma cholesterol and a greater risk of coronary artery disease. The Taconic apoE4 Targeted Replacement model has normal plasma cholesterol and triglyceride levels, but altered quantities of different plasma lipoprotein particles. This model also has delayed plasma clearance of cholesterol-rich lipoprotein particles (VLDL), with only half the clearance rate seen in the apoE3 Targeted Replacement model. Like the apoE3 model, the apoE4 mice develop altered plasma lipoprotein values and atherosclerotic plaques on an atherogenic diet. However, the atherosclerosis is more severe in the apoE4 model, with larger plaques and cholesterol apoE and apoB-48 levels twice that seen in the apoE3 model. The Taconic apoE4 Targeted Replacement model, along with the apoE2 and apoE3 Targeted Replacement Mice, provide an excellent tool for in vivo study of the human apoE isoforms.

CETP Transgenic Mice (Order Model #: 1003-T) These animals express the human plasma enzyme, CETP, resulting in mice with a dramatic reduction in serum HDL cholesterol. The mice can be useful in identifying and evaluating compounds that increase the levels of HDL cholesterol for reducing the risk of developing atherosclerosis

Transgene/Promoter: human apolipoprotein A-I These mice produce mouse HDL cholesterol particles that contain human apolipoprotein A-I. Transgenic expression is life-long in both sexes (Biochemical Genetics and Metabolism Laboratory, Rockefeller University, NY City).

A Mouse Model for Abetalipoproteinemia Abetalipoproteinemia, an inherited human disease characterized by a near-complete absence of apoB-containing lipoproteins in the plasma, is caused by mutations in the gene for microsomal triglyceride transfer protein (MTP). Gene targeting was used to knock out the mouse MTP gene (Mttp). In heterozygous knockout mice (Mttp^(+/−)), the MTP mRNA, protein, and activity levels were reduced by 50% in both liver and intestine. Recent studies with heterozygous MTP knockout mice have suggested that half-normal levels of MTP in the liver reduce apoB secretion. They hypothesized that reduced apoB secretion in the setting of half-normal MTP levels might be caused by a reduced MTP:apoB ratio in the endoplasmic reticulum, which would reduce the number of apoB-MTP interactions. If this hypothesis were true, half-normal levels of MTP might have little impact on lipoprotein secretion in the setting of half-normal levels of apoB synthesis (since the ratio of MTP to apoB would not be abnormally low) and might cause an exaggerated reduction in lipoprotein secretion in the setting of apoB overexpression (since the ratio of MTP to apoB would be even lower). To test this hypothesis, they examined the effects of heterozygous MTP deficiency on apoB metabolism in the setting of normal levels of apoB synthesis, half-normal levels of apoB synthesis (heterozygous Apob deficiency), and increased levels of apoB synthesis (transgenic overexpression of human apoB). Contrary to their expectations, half-normal levels of MTP reduced plasma apoB-100 levels to the same extent (˜25-35%) at each level of apoB synthesis. In addition, apoB secretion from primary hepatocytes was reduced to a comparable extent at each level of apoB synthesis. Thus, these results indicate that the concentration of MTP within the endoplasmic reticulum, rather than the MTP:apoB ratio, is the critical determinant of lipoprotein secretion. Finally, heterozygosity for an apoB knockout mutation was found to lower plasma apoB-100 levels more than heterozygosity for an MTP knockout allele. Consistent with that result, hepatic triglyceride accumulation was greater in heterozygous apoB knockout mice than in heterozygous MTP knockout mice. Cre/loxP tissue-specific recombination techniques were also used to generate liver-specific Mttp knockout mice. Inactivation of the Mttp gene in the liver caused a striking reduction in very low density lipoprotein (VLDL) triglycerides and large reductions in both VLDL/low density lipoproteins (LDL) and high density lipoprotein cholesterol levels. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Currently being tested is the hypothesis that accumulation of triglycerides in the liver renders the liver more susceptible to injury by a second insult (e.g., lipopolysaccharide).

Human apo B (apolipoprotein B) Transgene mice show apo B locus may have a causative role male infertility The fertility of apoB (apolipoprotein B) (+/−) mice was recorded during the course of backcrossing (to C57BL/6J mice) and test mating. No apparent fertility problem was observed in female apoB (+/−) and wild-type female mice, as was documented by the presence of vaginal plugs in female mice. Although apoB (+/−) mice mated normally, only 40% of the animals from the second backcross generation produced any offspring within the 4-month test period. Of the animals that produced progeny, litters resulted from <50% of documented matings. In contrast, all wild-type mice (6/6—i.e., 100%) tested were fertile. These data suggest genetic influence on the infertility phenotype, as a small number of male heterozygotes were not sterile. Fertilization in vivo was dramatically impaired in male apoB (+/−) mice. 74% of eggs examined were fertilized by the sperm from wild-type mice, whereas only 3% of eggs examined were fertilized by the sperm from apoB (+/−) mice. The sperm counts of apoB (+/−) mice were mildly but significantly reduced compared with controls. However, the percentage of motile sperm was markedly reduced in the apoB (+/−) animals compared with that of the wild-type controls. Of the sperm from apoB (+/−) mice, 20% (i.e., 4.9% of the initial 20% motile sperm) remained motile after 6 hr of incubation, whereas 45% (i.e., 33.6% of the initial 69.5%) of the motile sperm retained motility in controls after this time. In vitro fertilization yielded no fertilized eggs in three attempts with apo B (+/−) mice, while wild-type controls showed a fertilization rate of 53%. However, sperm from apoB (+/−) mice fertilized 84% of eggs once the zona pellucida had been removed. Numerous sperm from apoB (+/−) mice were seen binding to zona-intact eggs. However, these sperm lost their motility when observed 4-6 hours after binding, showing that sperm from apoB (+/−) mice were unable to penetrate the zona pellucida but that the interaction between sperm and egg was probably not direct. Sperm binding to zona-free oocytes was abnormal. In the apoB (+/−) mice, sperm binding did not attenuate, even after pronuclei had clearly formed, suggesting that apoB deficiency results in abnormal surface interaction between the sperm and egg.

Knockout of the mouse apoB gene resulted in embryonic lethality in homozygotes, protection against diet-induced hypercholesterolemia in heterozygotes, and developmental abnormalities in mice.

Model of insulin resistance, dyslipidemia & overexpression of human apoB It was shown that the livers of apoB mice assemble and secrete increased numbers of VLDL particles.

Example 3 Treatment of Diabetes Type-2 with iRNA

Introduction The regulation of hepatic gluconeogenesis is an important process in the adjustment of the blood glucose level. Pathological changes in the glucose production of the liver are a central characteristic in type-2-diabetes. For example, the fasting hyperglycemia observed in patients with type-2-diabetes reflects the lack of inhibition of hepatic gluconeogenesis and glycogenolysis due to the underlying insulin resistance in this disease. Extreme conditions of insulin resistance can be observed for example in mice with a liver-specific insulin receptor knockout (‘LIRKO’). These mice have an increased expression of the two rate-limiting gluconeogenic enzymes, phosphoenolpyruvate carboxykinase (PEPCK) and the glucose-6-phosphatase catalytic subunit (G6Pase). Insulin is known to repress both PEPCK and G6Pase gene expression at the transcriptional level and the signal transduction involved in the regulation of G6Pase and PEPCK gene expression by insulin is only partly understood. While PEPCK is involved in a very early step of hepatic gluconeogenesis (synthesis of phosphoenolpyruvate from oxaloacetate), G6Pase catalyzes the terminal step of both, gluconeogenesis and glycogenolysis, the cleavage of glucose-6-phosphate into phosphate and free glucose, which is then delivered into the blood stream.

The pharmacological intervention in the regulation of expression of PEPCK and G6Pase can be used for the treatment of the metabolic aberrations associated with diabetes. Hepatic glucose production can be reduced by an iRNA-based reduction of PEPCK and G6Pase enzymatic activity in subjects with type-2-diabetes.

Targets for iRNA

Glucose-6-phosphatase (G6Pase)

G6Pase mRNA is expressed principally in liver and kidney, and in lower amounts in the small intestine. Membrane-bound G6Pase is associated with the endoplasmic reticulum. Low activities have been detected in skeletal muscle and in astrocytes as well.

G6Pase catalyzes the terminal step in gluconeogenesis and glycogenolysis. The activity of the enzyme is several fold higher in diabetic animals and probably in diabetic humans. Starvation and diabetes cause a 2-3-fold increase in G6Pase activity in the liver and a 2-4-fold increase in G6Pase mRNA.

Phosphoenolpyruvate carboxykinase (PEPCK)

Overexpression of PEPCK in mice results in symptoms of type-2-diabetes mellitus. PEPCK overexpression results in a metabolic pattern that increases G6Pase mRNA and results in a selective decrease in insulin receptor substrate (IRS)-2 protein, decreased phosphatidylinositol 3-kinase activity, and reduced ability of insulin to suppress gluconeogenic gene expression.

TABLE 7 Other targets to inhibit hepatic glucose production Target Comment FKHR good evidence for antidiabetic phenotype (Nakae et al., Nat Genetics 32: 245(2002) Glucagon Glucagon receptor Glycogen phosphorylase PGC-1 (PPAR-Gamma regulates the cAMP response Coactivator) (and probably the PKB/FKHR- regulation) on PEPCK/G6Pase Fructose-1,6-bisphosphatase Glucose-6-phospate translocator Glucokinase inhibitory regulatory protein

Materials and Methods

Animals: BKS.Cg-m +/+ Lepr db mice, which contain a point mutation in the leptin receptor gene are used to examine the efficacy of iRNA for the targets listed above.

BKS.Cg-m +/+ Lepr db are available from the Jackson Laboratory (Stock Number 000642). These animals are obese at 3-4 weeks after birth, show elevation of plasma insulin at 10 to 14 days, elevation of blood sugar at 4 to 8 weeks, and uncontrolled rise in blood sugar. Exogenous insulin fails to control blood glucose levels and gluconeogenic activity increases.

The following numbers of male animals (age>12 weeks) would ideally be tested with the following iRNAs:

-   -   PEPCK, 2 sequences, 5 animals per sequence     -   G6Pase, 2 sequences, 5 animals per sequence     -   1 nonspecific sequence, 5 animals     -   1 control group (only injected, no siRNA), 5 animals     -   1 control group (not injected, no siRNA), 5 animals

Reagents: Necessary reagents would ideally include a Glucometer Elite XL (Bayer, Pittsburgh, Pa.) for glucose quantification, and an Insulin Radioimmunoassay (RIA) kit (Amersham, Piscataway, N.J.) for insulin quanitation

Assays:

G6P enzyme assays and PEPCK enzyme assays are used to measure the activity of the enzymes. Northern blotting is used to detect levels of G6Pase and PEPCK mRNA.

Antibody-based techniques (e.g., immunoblotting, immunofluorescence) are used to detect levels of G6Pase and PEPCK protein. Glycogen staining is used to detect levels of glycogen in the liver. Histological analysis is performed to analyze tissues.

Gene information:

G6Pase GenBank® No.: NM_008061,Mus musculus glucose-6-phosphatase, catalytic (G6pc), mRNA 1..2259, ORF 83 . . . 1156;

GenBank® No: U00445,Mus musculus glucose-6-phosphatase mRNA, complete cds 1..2259, ORF 83 . . . 1156

GenBank® No: BC013448

PEPCK

GenBank® No: NM 011044, Mus musculus phosphoenolpyruvate carboxykinase 1, cytosolic (Pck1), mRNA.1 . . . 2618, ORF 141 . . . 2009

GenBank® No: AF009605.1

Administration of iRNA:

iRNA corresponding to the genes described above would be administered to mice with hydrodynamic injection. One control group of animals would be treated with Metformin as a positive control for reduction in hepatic glucose levels.

Experimental Protocol

Mice would be housed in a facility in which there is light from 7:00 AM to 7:00 PM. Mice would be fed ad libidum from 7:00 PM to 7:00 AM and fast from 7:00 AM to 7:00 PM.

Day 0: 7:00 PM: Approximately 100 μl blood would be drawn from the tail. Serum would be isolated to measure glucose, insulin, HbA1c (EDTA-blood), glucagon, FFAs, lactate, corticosterone, serum triglycerides.

Day 1-7: Blood glucose would be measured daily at 8:00 AM and 6:00 PM (approx. 3-5 μl; measured with a Haemoglucometer)

Day 8: Blood glucose would be measured daily at 8:00 AM and 6:00 PM. iRNA would be injected between 10:00 AM and 2:00 PM

Day 9-20: Blood glucose would be measured daily at 8:00 AM and 6:00 PM.

Day 21: Mice would be sacrificed after 10 hours of fasting.

Blood would be isolated. Glucose, insulin, HbA1c (EDTA-blood), glucagon, FFAs, lactate, corticosterone, serum triglycerides would be measured. Liver tissue would be isolated for histology, protein assays, RNA assays, glycogen quantitation, and enzyme assays.

Example 4 Inhibition of Glucose-6-Phosphatase iRNA In Vivo

iRNA targeted to the Glucose-6-Phosphatase (G6P) gene was used to examine the effects of inhibition of G6P expression on glucose metabolism in vivo.

Female mice, 10 weeks of age, strain BKS.Cg-m +/+ Lepr db (The Jackson Laboratory) were used for in vivo analysis of enzymes of the hepatic glucose production. Mice were housed under conditions where it was light from 6:30 am to 6:30 pm. Mice were fed (ad libidum) during the night period and fasted during the day period.

On day 1, approximately 100 μl of blood was collected from test animals by puncturing the retroorbital plexus. On days 1-7, blood glucose was measured in blood obtained from tail veins (approximately 3-5 μl) using a Glucometer (Elite XL, Bayer). Blood glucose was sampled daily at 8 am and 6 μm.

On day 7 at approximately 2 μm, GL3 plasmid (10 μg) and siRNAs (100 μg G6Pase specific, Renilla nonspecific or no siRNA control) were delivered to animals using hydrodynamic coinj ection.

On day 8, GL3 expression was analyzed by injection of luceferin (3 mg) after anaesthesia with avertin and imaging. This was done to control for successful hydrodynamic delivery.

On days 8-10, blood glucose was measured in blood obtained from tail veins (approximately 3-5 ml) using a Glucometer (Elite XL, Bayer).

On day 10, mice were sacrificed after 10 hours of fasting. Blood and liver were isolated from sacrificed animals.

Results: Coinjection of GL3 plasmid and G6Pase iRNA (G6P4) reduced blood glucose levels for the short term. Coinjection of GL3 plasmid and Renilla nonspecific iRNA had no effect on blood glucose levels.

Example 5 Selected Palindromic Sequences

Tables 8-13 below provide selected palindromic sequences from the following genes: human ApoB, human glucose-6-phosphatase, rat glucose-6-phosphatase, β-catenin, and hepatitis C virus (HCV).

TABLE 8 Start End Start End Source Index Index Match Index Index # B SEQ ID NO: 1 ggccattccagaagggaag 509 528 SEQ ID NO: 1004 cttccgttctgtaatggcc 5795 5814 1 9 SEQ ID NO: 2 tgccatctcgagagttcca 4099 4118 SEQ ID NO: 1005 tggaactctctccatggca 10876 10995 1 8 SEQ ID NO: 3 catgtcaaacactttgtta 7068 7075 SEQ ID NO: 1006 taacaaattccttgacatg 7358 7377 1 8 SEQ ID NO: 4 tttgttataaatcttattg 7068 7087 SEQ ID NO: 1007 caataagatcaatagcaaa 8990 9009 1 8 SEQ ID NO: 5 tctggaaaaagggtcatga 8880 8699 SEQ ID NO: 1008 tccatgtcccatttacaga 11358 11375 1 8 SEQ ID NO: 6 cagctcttgttcaggtcca 10900 10919 SEQ ID NO: 1009 tggacctgcaccaaagctg 13952 13971 1 8 SEQ ID NO: 7 ggaggttccccagctctgc 356 375 SEQ ID NO: 1010 gcagccctgggaaaactcc 8447 6466 1 7 SEQ ID NO: 8 ctgttttgaagactctcca 1081 1100 SEQ ID NO: 1011 tggagggtagtcataacag 10327 10346 1 7 SEQ ID NO: 9 agtggctgaaacgtgtgca 1297 1316 SEQ ID NO: 1012 tgcagagctttctgccact 13508 13527 1 7 SEQ ID NO: 10 ccaaaatagaagggaatct 2068 2087 SEQ ID NO: 1013 agattcctttctttttcaa 4000 4019 1 7 SEQ ID NO: 11 tgaagagaagattgaattt 3620 3638 SEQ ID NO: 1014 aaattctcttttattttca 9212 9231 1 7 SEQ ID NO: 12 agtggtggcaacaccagca 4230 4249 SEQ ID NO: 1015 tgctagtgaggccaacact 10649 10668 1 7 SEQ ID NO: 13 aaggctccacaagtcatca 5950 5989 SEQ ID NO: 1016 tgatgatatctggaacctt 10724 10743 1 7 SEQ ID NO: 14 gtccgccaggtttctagca 7725 7744 SEQ ID NO: 1017 tgctaagaaccttactgac 7781 7800 1 7 SEQ ID NO: 15 tgatatctggaaccttgga 10727 10745 SEQ ID NO: 1018 ttcactgttcctgaaatca 7863 7882 1 7 SEQ ID NO: 16 gtcaagttgagcaatttct 13423 13442 SEQ ID NO: 1019 agaaaaggcacaccttgac 11072 11091 1 7 SEQ ID NO: 17 atccagatggaaaagggaa 13480 13499 SEQ ID NO: 1020 ttccaatttccctgtggat 3680 3698 1 7 SEQ ID NO: 18 atttgtttgtcaaagaagt 4543 4562 SEQ ID NO: 1021 acttcagagagaaatacat 11401 11420  4 6 SEQ ID NO: 19 ctggaaaatgtcagcctgg 204 223 SEQ ID NO: 1022 ccagacttcagttaccagc 8235 8264 2 6 SEQ ID NO: 20 accaggaggttcttcttca 1729 1748 SEQ ID NO: 1023 tgaagtgtgatctcctggt 5089 5108 2 6 SEQ ID NO: 21 aaagaagttctgaaaagat 1956 1975 SEQ ID NO: 1024 attccatcacaaaatcctt 9661 9680 2 6 SEQ ID NO: 22 gctacagcttatggctcca 3570 3589 SEQ ID NO: 1025 tggatctaaatgcagtagc 11623 11642 2 6 SEQ ID NO: 23 atcaatatgatcaatttgt 6414 3433 SEQ ID NO: 1026 caaagaaatcaagattgat 4553 4572 2 6 SEQ ID NO: 24 gaattatctttaaaacatt 7326 7345 SEQ ID NO: 1027 atgtggacaaatataccgg 11494 11513 2 6 SEQ ID NO: 25 cgaggcccgcgctgctggc 130 149 SEQ ID NO: 1028 gccagaagtgagatcctcg 3507 3526 1 6 SEQ ID NO: 26 acaactatgaggctgagag 271 290 SEQ ID NO: 1029 ctctgagcaacaaattttt 10309 10326 1 6 SEQ ID NO: 27 gctgagagttccagtggag 282 301 SEQ ID NO: 1030 ctccatggcaaatgtcagg 10885 10904 1 6 SEQ ID NO: 28 tgaagaaaaaccaagaact 448 467 SEQ ID NO: 1031 gagtcattgaggttcttca 4929 494 1 6 SEQ ID NO: 29 cctacttacatcctgaaca 558 577 SEQ ID NO: 1032 tgttcataagggaggtagg 12766 12785 1 6 SEQ ID NO: 30 ctacttacatcctgaacat 559 578 SEQ ID NO: 1033 atgttcataagggaggtag 12785 12784 1 6 SEQ ID NO: 31 gagacagaagccaagagcc 615 634 SEQ ID NO: 1034 gcttggttttgcccagtct 2459 2478 1 6 SEQ ID NO: 32 cactcactttaccgtcaag 671 690 SEQ ID NO: 1035 cttgaacaccaaagtcact 6000 6019 1 6 SEQ ID NO: 33 ctgatcagcagcagccagt 822 641 SEQ ID NO: 1036 actgggaagtgcttatcag 5237 5256 1 6 SEQ ID NO: 34 actggacgctaagaggaag 854 873 SEQ ID NO: 1037 cttccccaaagagaggagt 2890 2909 1 6 SEQ ID NO: 35 agaggaagcatgtggcaga 865 884 SEQ ID NO: 1038 tctggcatttactttctct 5921 5940 1 6 SEQ ID NO: 36 tgaagactctccaggaact 1087 1106 SEQ ID NO: 1039 agttgaaggagactattca 7216 7235 1 6 SEQ ID NO: 37 ctctgagcaaaatatccag 1121 1140 SEQ ID NO: 1040 ctggttactgagctgagag 1151 1180 1 6 SEQ ID NO: 38 atgaagcagtcacatctct 1189 1208 SEQ ID NO: 1041 agagctgccagtcttcatt 10016 10035 1 6 SEQ ID NO: 39 ttgccacagctgattgagg 1209 1228 SEQ ID NO: 1042 cctcctacagtggtggcaa 4222 4241 1 6 SEQ ID NO: 40 agctgattgaggtgtccag 1216 1235 SEQ ID NO: 1043 ctggattccacatgcagct 11847 11866 1 6 SEQ ID NO: 41 tgctccactcacatcctcc 1276 1297 SEQ ID NO: 1044 ggaggctttaagttcagca 7601 7620 1 6 SEQ ID NO: 42 tgaaacgtgtgcatgtcaa 1303 1322 SEQ ID NO: 1045 ttgggagagacaagtttca 6500 6519 1 6 SEQ ID NO: 43 gacattgctaattacctga 1503 1522 SEQ ID NO: 1046 tcagaagctaagcaatgtc 7282 7251 1 6 SEQ ID NO: 44 ttcttcttcagactttcct 1735 1757 SEQ ID NO: 1047 aggagagtccaaatttaga 8498 8517 1 6 SEQ ID NO: 45 ccaatatcttgaaactcag 1903 1922 SEQ ID NO: 1048 tctgaattcattcaattgg 8485 6504 1 6 SEQ ID NO: 46 aaagttagtgaaaagaagt 1946 1965 SEQ ID NO: 1049 aactaccctcactgccttt 2132 2151 1 6 SEQ ID NO: 47 aagttagtgaaagaagttc 1947 1966 SEQ ID NO: 1050 gaacctctggcattttact 5918 5935 1 6 SEQ ID NO: 48 aaagaagttctgaaagaat 1956 1975 SEQ ID NO: 1051 attctctggtaactacttt 5482 5501 1 6 SEQ ID NO: 49 tttggctataccaaagatg 2322 2341 SEQ ID NO: 1052 catcttaggcactgacaaa 4997 5016 1 6 SEQ ID NO: 50 tgttgagaagctgattaaa 2381 2400 SEQ ID NO: 1053 tttagccatcggctcaaca 5700 5719 1 6 SEQ ID NO: 51 caggaagggctcaaagaat 2561 2580 SEQ ID NO: 1054 attcctttaacaattcctg 9492 9511 1 6 SEQ ID NO: 52 aggaagggctcaaagaatg 2562 2581 SEQ ID NO: 1055 cattcctttaacaattcct 9491 9510 1 6 SEQ ID NO: 53 gaagggctcaaagaatgac 2564 2583 SEQ ID NO: 1056 gtcagtcttcaggctcttc 7914 7933 1 6 SEQ ID NO: 54 caaagaatgacttttttct 2572 2591 SEQ ID NO: 1057 agaaggatggcattttttg 14000 14019 1 6 SEQ ID NO: 55 catggagaatgcctttgaa 2603 2622 SEQ ID NO: 1058 ttcagagccaaagtccatg 7119 7138 1 6 SEQ ID NO: 56 ggagccaaggctggagtaa 2679 2698 SEQ ID NO: 1059 ttactccaacgccagctcc 3050 3069 1 6 SEQ ID NO: 57 tcattccttccccaaagag 2884 2903 SEQ ID NO: 1060 ctctctggggcatctatgc 5139 5155 1 6 SEQ ID NO: 58 acctatgagctccagagag 3165 3184 SEQ ID NO: 1061 ctctcaagaccacagaagg 12976 12995 1 6 SEQ ID NO: 59 gggcaaaaacgtcttacag 3355 3384 SEQ ID NO: 1062 tctgaaagacaacgtgccc 12317 12336 1 6 SEQ ID NO: 60 accctggaccttcagaaca 3387 3406 SEQ ID NO: 1063 tgttgctaaggttcagggt 5675 5694 1 6 SEQ ID NO: 61 atgggcgacctaagttgtg 3429 3448 SEQ ID NO: 1064 cacaaattagtttcaccat 8941 8960 1 6 SEQ ID NO: 62 gatgaagagaagattgaat 3618 3637 SEQ ID NO: 1065 atccagcttccccacactt 8330 8349 1 6 SEQ ID NO: 63 caatgtgataccaaaaaaa 3556 3675 SEQ ID NO: 1066 tttttggaaatgccattgt 8643 8662 1 6 SEQ ID NO: 64 gtagataccaaaaaaatga 3660 3679 SEQ ID NO: 1067 tcatgtgatgggtctctac 4371 4390 1 6 SEQ ID NO: 65 gcttcagttcatttggact 4509 4528 SEQ ID NO: 1068 agtcaagaaggacttaagc 5304 5323 1 6 SEQ ID NO: 66 tttgtttgtcaaagaagtc 4544 4563 SEQ ID NO: 1069 gacttcagagaaatacaaa 11400 11419 1 6 SEQ ID NO: 67 ttgtttgtcaaagaagtca 4545 4564 SEQ ID NO: 1070 tgacttcagagaaatacaa 11399 11418 1 6 SEQ ID NO: 68 tggcaatgggaaactcgct 5846 5865 SEQ ID NO: 1071 agcgagagtcccctgccat 8219 8238 1 6 SEQ ID NO: 69 aacctctggcatttacttt 5917 5936 SEQ ID NO: 1072 aaaggagatgtcaagggtt 10599 10618 1 6 SEQ ID NO: 70 catttactttctctcatga 5926 5945 SEQ ID NO: 1073 tcatttgaaagaataaatg 7026 7045 1 6 SEQ ID NO: 71 aaagtcagtgccctgctta 6009 6028 SEQ ID NO: 1074 taagaaccttactgacttt 7784 7803 1 6 SEQ ID NO: 72 tcccattttttgagacctt 6322 6341 SEQ ID NO: 1075 aaggacttcaggaatggga 12004 12023 1 6 SEQ ID NO: 73 catcaatattgatcaattt 6413 6432 SEQ ID NO: 1076 aaattaaaaagtcttgatg 6732 6751 1 6 SEQ ID NO: 74 taaagatagttatgattta 6665 6684 SEQ ID NO: 1077 taaaccaaaaacttggtta 9019 9038 1 6 SEQ ID NO: 75 tattgatgaaatcattgaa 6713 6732 SEQ ID NO: 1078 ttcaaagacttaaaaaata 8007 8026 1 6 SEQ ID NO: 76 atgatctacatttgtttat 6790 5809 SEQ ID NO: 1079 ataaagaaattaaagtcat 7380 7399 1 6 SEQ ID NO: 77 agagacacatacagaatat 6919 6936 SEQ ID NO: 1080 atatattgtcagtgcctct 13385 13401 1 6 SEQ ID NO: 78 gacacatacagaatataga 6922 6941 SEQ ID NO: 1081 tctaaattcagttcttgtc 11327 11349 1 6 SEQ ID NO: 79 agcatgtcaaacactttgt 7054 7073 SEQ ID NO: 1082 acaaagtcagtgccctgct 6007 8026 1 6 SEQ ID NO: 80 tttttagaggaaaccaagg 7515 7534 SEQ ID NO: 1083 ccttgtgtacaccaaaaac 11230 11249 1 6 SEQ ID NO: 81 ttttagaggaaaccaaggc 7516 7535 SEQ ID NO: 1084 gcctttgtgtacaccaaaa 11229 11248 1 6 SEQ ID NO: 82 ggaagatagacttcctgaa 9307 9326 SEQ ID NO: 1085 ttcagaaatactgtttttc 12824 12843 1 6 SEQ ID NO: 83 cactgtttctgagtcccag 9334 9353 SEQ ID NO: 1086 ctgggacctaccaagagtg 12523 12542 1 6 SEQ ID NO: 84 cacaaatcctttggctgtg 9668 9687 SEQ ID NO: 1087 cacatttcaaggaattgtg 10063 10082 1 6 SEQ ID NO: 85 ttcctggatacactgttcc 9853 9872 SEQ ID NO: 1088 ggaactgttgactcaggac 12569 12588 1 6 SEQ ID NO: 86 gaaatctcaagctttctct 10042 10061 SEQ ID NO: 1089 agagccaggtcgagctttc 11044 11063 1 6 SEQ ID NO: 87 tttcttcatcttcatctgt 10210 10229 SEQ ID NO: 1090 acagctgaaagagatgaaa 13055 13074 1 6 SEQ ID NO: 88 tctaccgctaaaggagcag 10521 10540 SEQ ID NO: 1091 ctgcacgctttgaggtaga 11761 11780 1 6 SEQ ID NO: 89 ctaccgctaaaggagcagt 10522 10541 SEQ ID NO: 1092 actgcacgctttgaggtcg 11760 11779 1 6 SEQ ID NO: 90 aggggcctcttttcaccaa 10631 10650 SEQ ID NO: 1093 ttggccaggaagtggccct 10957 10976 1 6 SEQ ID NO: 91 tctccatccctgtaaaaag 11285 11284 SEQ ID NO: 1094 ctttttcaccaacggagaa 10838 10857 1 6 SEQ ID NO: 92 gaaaaacaaagcagattat 11816 11635 SEQ ID NO: 1095 ataaactgcaagatttttc 13600 13619 1 6 SEQ ID NO: 93 actcactcattgattttct 12682 12701 SEQ ID NO: 1096 agaaaatcaggatcgtagt 14027 14046 1 6 SEQ ID NO: 94 taaactaatagatgtaatc 12890 12909 SEQ ID NO: 1097 gattaccaccagcagttta 13578 13597 1 6 SEQ ID NO: 95 caaaaacgagcttcggaag 13200 13219 SEQ ID NO: 1098 cttcgtgagaatatttgaa 13260 13279 1 6 SEQ ID NO: 96 tggaataatgctcagtgtt 2366 2385 SEQ ID NO: 1099 aacacttaacttgaattca 10562 10681 3 5 SEQ ID NO: 97 gatttgaaatccaaagaag 2400 2419 SEQ ID NO: 1100 cttcagagaaatacaaatc 11402 11421 3 5 SEQ ID NO: 98 atttgaaatccaaagaagt 2401 2420 SEQ ID NO: 1101 acttcagagaaatacaaat 11401 11420 3 5 SEQ ID NO: 99 atcaacagccgcttctttg 990 1009 SEQ ID NO: 1102 caaagaagtcaagattgat 4553 4572 2 5 SEQ ID NO: 100 tgttttgaagactctccag 1082 1101 SEQ ID NO: 1103 ctggaaagttaaaacaaac 6955 6974 2 5 SEQ ID NO: 101 cccttctgatagatgtggt 1324 1343 SEQ ID NO: 1104 accaaagctggcaccaggg 13961 13980 2 5 SEQ ID NO: 102 tgagcaagtgaagaacttt 1868 1887 SEQ ID NO: 1105 aaagccattcagtctctca 12963 12982 2 5 SEQ ID NO: 103 atttgaaatccaaagaagt 2401 2420 SEQ ID NO: 1106 actttctaaacttgaaatt 9055 9074 2 5 SEQ ID NO: 104 atccaaagaagtcccggaa 2408 227 SEQ ID NO: 1107 tccggggaaacctgggatt 12721 12740 2 5 SEQ ID NO: 105 agagcctacctccgcatct 2430 2449 SEQ ID NO: 1108 agatggtacgttagcctct 11921 11940 2 5 SEQ ID NO: 106 aatgcctttgaactcccca 2610 2629 SEQ ID NO: 1109 tgggaactacaattcattt 7012 7031 2 5 SEQ ID NO: 107 gaagtccaaattccggatt 3297 3316 SEQ ID NO: 1110 aatcttcaatttattcttc 13815 13834 2 5 SEQ ID NO: 108 tgcaagcagaagccagaag 3496 3515 SEQ ID NO: 1111 cttcaggttccatcgtgca 11376 11395 2 5 SEQ ID NO: 109 gaagagaagattgaatttg 3621 3640 SEQ ID NO: 1112 caaaacctactgtgcttcc 10459 10476 2 5 SEQ ID NO: 110 atgctaaaggcacatatgg 4597 4616 SEQ ID NO: 1113 ccatatgaaagtcaagcat 12656 12675 2 5 SEQ ID NO: 111 tccctcacctccacctctg 4737 4756 SEQ ID NO: 1114 cagattctcagatgaggga 8912 8931 2 5 SEQ ID NO: 112 atttacagctctgacaagt 5427 5446 SEQ ID NO: 1115 acttttctaaacttgaaat 9055 9074 2 5 SEQ ID NO: 113 aggagcctaccaaaataat 5594 5513 SEQ ID NO: 1116 attatgttgaaaacagtct 11830 11849 2 5 SEQ ID NO: 114 aaagctgaagcacatcaat 6401 8420 SEQ ID NO: 1117 attgttgctcatctccttt 10184 10213 2 5 SEQ ID NO: 115 ctgctggaaacaacgagaa 9416 9437 SEQ ID NO: 1118 ttctgattaccaccagcag 13574 13593 2 5 SEQ ID NO: 116 ttgaaggaattcttgaaaa 9582 9601 SEQ ID NO: 1119 ttttaaaagaaatcttcaa 13805 13824 2 5 SEQ ID NO: 117 gaagtaaaagaaaattttg 10743 10762 SEQ ID NO: 1120 caaaacctactgtctcttc 10459 10476 2 5 SEQ ID NO: 118 tgaagaagatggcaaattt 11984 12003 SEQ ID NO: 1121 aaatgtcagctcttgttca 10894 10913 2 5 SEQ ID NO: 119 aggatctgagttatttttc 14035 14054 SEQ ID NO: 1122 gcaagtcagcccagttcct 10920 10939 2 5 SEQ ID NO: 120 gtgcccttctcggttgctg 18 37 SEQ ID NO: 1123 cagccattgacatgagcac 5740 5759 1 5 SEQ ID NO: 121 ggcgcggcctgcgctgctg 146 165 SEQ ID NO: 1124 cagctccacagactccgcc 3062 3061 1 5 SEQ ID NO: 122 ctgcgctgctgctgctgct 154 173 SEQ ID NO: 1125 agcagaaggtgcgaagcag 3224 3243 1 5 SEQ ID NO: 123 gctggtggcgggcgccagg 170 189 SEQ ID NO: 1126 cctggattccacatgcagc 11646 11865 1 5 SEQ ID NO: 124 aagaggaaatgctggaaaa 193 212 SEQ ID NO: 1127 tttttcttcactacatctt 2584 2603 1 5 SEQ ID NO: 125 ctggaaaatgtcagcctgg 204 223 SEQ ID NO: 1128 ccagacttccacatcccag 3915 3934 1 5 SEQ ID NO: 126 tggagtccctgggactgct 296 315 SEQ ID NO: 1129 agcatgcctagtttctcca 9945 9964 1 5 SEQ ID NO: 127 ggagtccctgggactgctg 297 316 SEQ ID NO: 1130 cagcatgcctagtttctcc 9944 9953 1 5 SEQ ID NO: 128 tgggactgctgattcaaga 305 324 SEQ ID NO: 1131 tcttccatcacttgaccca 2042 2061 1 5 SEQ ID NO: 129 ctgctgattcaagaagtgc 310 329 SEQ ID NO: 1132 gcacaccttgacattgcag 11079 11098 1 5 SEQ ID NO: 130 tgccaccaggatcaactgc 326 345 SEQ ID NO: 1133 gcaggctgaactggtggca 2717 2736 1 5 SEQ ID NO: 131 gccaccaggatcaactgca 327 346 SEQ ID NO: 1134 tgcaggctgaactggtggc 2715 2735 1 5 SEQ ID NO: 132 tgcaaggttgagcgtggag 342 361 SEQ ID NO: 1135 cctccatcctctgatctga 4744 4763 1 5 SEQ ID NO: 133 caaggttgagctggaggtt 344 363 SEQ ID NO: 1136 aacccctacatgaagcttg 13755 13774 1 5 SEQ ID NO: 134 ctctgcagcttcatcctga 369 366 SEQ ID NO: 1137 tcaggaagcttctcaagag 13211 13230 1 5 SEQ ID NO: 135 cagcttcatcctgaagaac 374 393 SEQ ID NO: 1138 ggtcttgagttaaatgctg 4977 4996 1 5 SEQ ID NO: 136 gcttcatcctgaagaccag 376 395 SEQ ID NO: 1139 ctggacgctaagaggaagc 855 874 1 5 SEQ ID NO: 137 tcatcctgaagaccagcca 379 398 SEQ ID NO: 1140 tggcatggcattatgatga 3604 3623 1 5 SEQ ID NO: 138 gaaaaccaagaactctgag 452 471 SEQ ID NO: 1141 ctcaaccttaatgattttc 8286 8305 1 5 SEQ ID NO: 139 agaactctgaggagtttgc 460 479 SEQ ID NO: 1142 gcaagctatatcagtattc 8377 8396 1 5 SEQ ID NO: 140 tctgaggagtttgctgcag 465 484 SEQ ID NO: 1143 ctgcagggatcccccagat 2528 2545 1 5 SEQ ID NO: 141 tttgctgcagccatgtcca 474 493 SEQ ID NO: 1144 tggaagtgtcagtggcaaa 10372 10391 1 5 SEQ ID NO: 142 caagaggggcatcatttct 578 597 SEQ ID NO: 1145 agaataaatgacgttcttg 7035 7054 1 5 SEQ ID NO: 143 tcactttaccgtcaagacg 674 693 SEQ ID NO: 1146 cgtctacactatcatgtga 4360 4379 1 5 SEQ ID NO: 144 tttaccgtcaagacgagga 678 697 SEQ ID NO: 1147 tccttgacatgttgataaa 7366 7335 1 5 SEQ ID NO: 145 cactggacgctaagaggaa 653 672 SEQ ID NO: 1148 ttccagaaagcagccagtg 12498 12517 1 5 SEQ ID NO: 146 aggaagcatgtggcagaag 867 886 SEQ ID NO: 1149 cttcatacacattaatcct 9988 10007 1 5 SEQ ID NO: 147 caaggagcaacacctcttc 893 912 SEQ ID NO: 1150 gaagtagtactgcatgttg 6835 6854 1 5 SEQ ID NO: 148 acagactttgaaacttgaa 959 978 SEQ ID NO: 1151 ttcaattcttcaatgctgt 10500 10519 1 5 SEQ ID NO: 149 tgatgaagcagtcacatct 1187 1206 SEQ ID NO: 1152 agatttgaggattccatca 7975 7995 1 5 SEQ ID NO: 150 agcagtcacatctctcttg 1193 1212 SEQ ID NO: 1153 caaggagaaactgactcgt 6524 6543 1 5 SEQ ID NO: 151 ccagccccatcactttaca 1231 1250 SEQ ID NO: 1154 tgtagtctcctggtgctgg 5094 5113 1 5 SEQ ID NO: 152 ctccactccatcctccagg 1280 1299 SEQ ID NO: 1155 ctggagcttagtaatggag 8709 8728 1 5 SEQ ID NO: 153 catgccaacccccttctga 1314 1333 SEQ ID NO: 1156 tcagatgagggaacacatg 8919 8936 1 5 SEQ ID NO: 154 gagagatcttcaacatggc 1390 1409 SEQ ID NO: 1157 gccaccctggaactctctc 10859 10888 1 5 SEQ ID NO: 155 tcaacatggcgagggatca 1398 1418 SEQ ID NO: 1158 tgatcccacctctcattga 2965 2984 1 5 SEQ ID NO: 156 ccaccttgtatgcgctgag 1429 1446 SEQ ID NO: 1159 ctcagggatctgaaggtgg 8187 8205 1 5 SEQ ID NO: 157 gtcaacaactatcataaga 1455 1474 SEQ ID NO: 1160 tcttgagttaaatgctgac 4979 4998 1 5 SEQ ID NO: 158 tggacattgctaattacct 1501 1520 SEQ ID NO: 1161 aggtatattcgaaagtcca 12799 12618 1 5 SEQ ID NO: 159 ggacattgctaattacctg 1502 1521 SEQ ID NO: 1162 caggtatattcgaaagtcc 12798 12817 1 5 SEQ ID NO: 160 ttctgcgggtcattggaaa 1573 1592 SEQ ID NO: 1163 tttcacatgccaaggagaa 6514 6533 1 5 SEQ ID NO: 161 ccagaactcaagtcttcaa 1520 1639 SEQ ID NO: 1164 ttgaagtgtatgtctcctg 5088 5107 1 5 SEQ ID NO: 162 agtcttcaatcctgaaatg 1630 1649 SEQ ID NO: 1165 catttctgattggtggact 7757 7776 1 5 SEQ ID NO: 163 tgagcaagtgaagaacttt 1868 1887 SEQ ID NO: 1166 aaagtgccacttttactca 6183 6202 1 5 SEQ ID NO: 164 agcaagtgaagaactttgt 1870 1889 SEQ ID NO: 1167 acaaagtcagtgccctgct 6007 6026 1 5 SEQ ID NO: 165 tctgaaagaatctcaactt 1964 1983 SEQ ID NO: 1168 aagtccataatggttcaga 12611 12630 1 5 SEQ ID NO: 166 actgtcatggacttcagaa 1986 2005 SEQ ID NO: 1169 ttctgaatatattgtcagt 13376 13395 1 5 SEQ ID NO: 167 acttgacccagcctcagcc 2051 2070 SEQ ID NO: 1170 ggctcaccctgagagaagt 12391 12410 1 5 SEQ ID NO: 168 tccaaataactaccttcct 2096 2115 SEQ ID NO: 1171 aggaagatatgaagatgga 4712 4731 1 5 SEQ ID NO: 169 actaccctcactgcctttg 2133 2152 SEQ ID NO: 1172 caaatttgtggagggtagt 10319 10338 1 5 SEQ ID NO: 170 ttggatttgcttcagctga 2149 2168 SEQ ID NO: 1173 tcagtataagtacaaccaa 9392 9411 1 5 SEQ ID NO: 171 ttggaagctctttttggga 2211 2230 SEQ ID NO: 1174 tcccgattcacgcttccaa 11577 11596 1 5 SEQ ID NO: 172 ggaagctctttttgggaag 2213 2232 SEQ ID NO: 1175 cttcagaaagctaccttcc 7929 7946 1 5 SEQ ID NO: 173 tttttcccagacagtgtca 2238 2257 SEQ ID NO: 1176 tgaccttctctaagcaaaa 4876 4805 1 5 SEQ ID NO: 174 agacagtgtcaacaaagct 2246 2285 SEQ ID NO: 1177 agcttggttttgccagtct 2458 2477 1 5 SEQ ID NO: 175 ctttggctataccaaagat 2321 2340 SEQ ID NO: 1178 atctcgtgtctaggaaaag 5968 5987 1 5 SEQ ID NO: 176 caaagatgataaacatgag 2333 2382 SEQ ID NO: 1179 ctcaaggataacgtgtttg 12609 12628 1 5 SEQ ID NO: 177 gatatggtaaatggaataa 2356 2374 SEQ ID NO: 1180 ttatcttataattatatca 13079 13098 1 5 SEQ ID NO: 178 ggaataatgctcagtgttg 2367 2386 SEQ ID NO: 1181 caaacacttacttgaattc 10661 10680 1 5 SEQ ID NO: 179 tttgaaatccaaagaagtc 2402 2421 SEQ ID NO: 1182 gacttcagagaaatacaaa 11400 11419 1 5 SEQ ID NO: 180 gatcccccagatgattgga 2534 2553 SEQ ID NO: 1183 tccaatttccctgtggatc 3681 3700 1 5 SEQ ID NO: 181 cagatattggagaggtcaa 2541 2560 SEQ ID NO: 1184 tgaccacacaaaacagtct 5363 5382 1 5 SEQ ID NO: 182 agaatgacttttttctctc 2575 2594 SEQ ID NO: 1185 tgaagtccggattcattct 11015 11034 1 5 SEQ ID NO: 183 gaactccccactggagctg 2619 2638 SEQ ID NO: 1186 cagctcaaccgtacagttc 11851 11880 1 5 SEQ ID NO: 184 atatcttcatgggagtcaa 2652 2671 SEQ ID NO: 1187 tgacttcagtgcagaatat 11966 11965 1 5 SEQ ID NO: 185 gtcatgctccccggaggca 2657 2688 SEQ ID NO: 1188 tggccccgtttaccatgac 5809 5828 1 5 SEQ ID NO: 186 gctgaagtttatcattcct 2873 2892 SEQ ID NO: 1189 aggaggctttaagttcagc 7600 7619 1 5 SEQ ID NO: 187 attccttccccaaagagac 2886 2905 SEQ ID NO: 1190 gtctcttcctccatggaat 10470 10489 1 5 SEQ ID NO: 188 ctcattgagaacaggcagt 2976 2995 SEQ ID NO: 1191 actgactgcacgctttgag 11756 11775 1 5 SEQ ID NO: 189 ttgagcagtattctgtcag 3142 3161 SEQ ID NO: 1192 cttagagaagtgtcttcaa 12399 12418 1 5 SEQ ID NO: 190 accttgtccagtgaagtcc 3285 3304 SEQ ID NO: 1193 ggacggtactgtcccaggt 12784 12803 1 5 SEQ ID NO: 191 ccagtgaagtccaaattcc 3292 3311 SEQ ID NO: 1194 ggaaggcagagtttactgg 9148 9167 1 5 SEQ ID NO: 192 acattcagaacaagaaaat 3394 3413 SEQ ID NO: 1195 atttcctaaagctggatgc 11167 11186 1 5 SEQ ID NO: 193 gaaaaatcaagggtgttat 3463 3482 SEQ ID NO: 1196 ataaactgcaagatttttc 13600 13619 1 5 SEQ ID NO: 194 aaatcaagggtgttatttc 3466 3485 SEQ ID NO: 1197 gaaacaatgcattagattt 9745 9764 1 5 SEQ ID NO: 195 tggcattatgatgaagaga 3609 3628 SEQ ID NO: 1198 tctcccgtgtataatgcca 11781 11800 1 5 SEQ ID NO: 196 aagagaagattgaatttga 3622 3641 SEQ ID NO: 1199 tcaaaacctactgtctcct 10458 10477 1 5 SEQ ID NO: 197 aaatgacttccaatttccc 3673 3692 SEQ ID NO: 1200 gggaactacaatttcattt 7013 7032 1 5 SEQ ID NO: 198 atgacttccaatttccctg 3675 3694 SEQ ID NO: 1201 caggctgattacgagtcat 4917 4936 1 5 SEQ ID NO: 199 acttccaatttccctgtgg 3678 3697 SEQ ID NO: 1202 ccacgaaaaatatggaagt 10360 10379 1 5 SEQ ID NO: 200 agttgcaatgagctcatgg 3803 3822 SEQ ID NO: 1203 ccatcagttcagataaact 7969 8000 1 5 SEQ ID NO: 201 tttgcaagaccacctcaat 3860 3879 SEQ ID NO: 1204 attgacctgtccattcaaa 13671 13690 1 5 SEQ ID NO: 202 gaaggagttcaacctccag 388 3903 SEQ ID NO: 1205 ctggaattgtcattccttc 11728 11747 1 5 SEQ ID NO: 203 acttccacatcccagaaaa 43919 3938 SEQ ID NO: 1206 ttttaacaaaaagtggaag 6821 6840 1 5 SEQ ID NO: 204 ctcttcttaaaaagcgatg 3939 3956 SEQ ID NO: 1207 catcactgccaaaggagag 8486 8505 1 5 SEQ ID NO: 205 aaaagcgatggccgggtcc 3948 3967 SEQ ID NO: 1208 tgactcactcattgatttt 12680 12689 1 5 SEQ ID NO: 206 ttcctttgccttttggtgg 4003 4022 SEQ ID NO: 1209 ccacaaaacaatgaaggga 9256 9275 1 5 SEQ ID NO: 207 caagtctgtgggattccat 4079 4098 SEQ ID NO: 1210 atgggaaaaaacaggcttg 9566 9585 1 5 SEQ ID NO: 208 aagtccctacttttaccat 4117 4136 SEQ ID NO: 1211 atgggaagtataagaactt 4834 4853 1 5 SEQ ID NO: 209 tgcctctcctgggtgttct 4159 4176 SEQ ID NO: 1212 agaaaaacaaacaaacgga 9643 9662 1 5 SEQ ID NO: 210 accagcacagaccatttca 4242 4251 SEQ ID NO: 1213 tgaagtgtagtctcctggt 5089 5108 1 5 SEQ ID NO: 211 ccagcacagaccatttcag 4243 4262 SEQ ID NO: 1214 ctgaaatacaatgctctgg 5511 5530 1 5 SEQ ID NO: 212 actatcatgtgatgggtct 4367 4388 SEQ ID NO: 1215 agacacctgattttatagt 7948 7967 1 5 SEQ ID NO: 213 accacagatgtctgcttca 4498 4515 SEQ ID NO: 1216 tgaaggctgactctgtggt 4282 4301 1 5 SEQ ID NO: 214 ccacagagtgtctgcttcg 4497 4516 SEQ ID NO: 1217 ctgagcaacaatttgtgga 10311 10330 1 5 SEQ ID NO: 215 tttggactccaaaaagaaa 4520 4539 SEQ ID NO: 1218 tttctctcatgattacaaa 5933 5952 1 5 SEQ ID NO: 216 tcaaagaagtcaagattga 4552 4571 SEQ ID NO: 1219 tcaaggataactgtttgag 12610 12629 1 5 SEQ ID NO: 217 atgagaactacgagctgac 4798 4817 SEQ ID NO: 1220 gtcagatattgttgctcat 10167 10206 1 5 SEQ ID NO: 218 ttaaactctgacaccaatg 4818 4837 SEQ ID NO: 1221 cattcattgaagatgttaa 7342 7361 1 5 SEQ ID NO: 219 gaagtataagaactttgcc 4838 4857 SEQ ID NO: 1222 ggcaaatttgaaggacttc 11994 12013 1 5 SEQ ID NO: 220 aagtataagaaactttgca 4839 4858 SEQ ID NO: 1223 tggcaaatttgaaggactt 11993 12012 1 5 SEQ ID NO: 221 ttcttcagcctgctttctg 4941 4960 SEQ ID NO: 1224 cagaatccagatacaagaa 6664 6903 1 5 SEQ ID NO: 222 ctggatcactaaattccca 4957 4976 SEQ ID NO: 1225 tgggtctttccagagcccg 11033 11052 1 5 SEQ ID NO: 223 aaattaatagtggtgctca 5014 5033 SEQ ID NO: 1226 tgagaagccccaagaattt 6245 6267 1 5 SEQ ID NO: 224 ggccattccagaagggaag 5073 5092 SEQ ID NO: 1227 cttccgttctgtaatggcc 9848 9867 1 5 SEQ ID NO: 225 tgccatctcgagagttcca 5238 5257 SEQ ID NO: 1228 tggaactctctccatggca 8310 8329 1 5 SEQ ID NO: 226 catgtcaaacactttgtta 5278 5297 SEQ ID NO: 1229 taacaaattccttgacatg 10744 10763 1 5 SEQ ID NO: 227 tttgttataaatcttattg 5280 5299 SEQ ID NO: 1230 caataagatcaatagcaaa 10742 10761 1 5 SEQ ID NO: 228 tctggaaaaagggtcatga 5302 5321 SEQ ID NO: 1231 tccatgtcccatttacaga 13363 13382 1 5 SEQ ID NO: 229 cagctcttgttcaggtcca 5325 5344 SEQ ID NO: 1232 tggacctgcaccaaagctg 6205 6224 1 5 SEQ ID NO: 230 ggaggttccccagctctgc 5367 5388 SEQ ID NO: 1233 gcagccctgggaaaactcc 11219 11238 1 5 SEQ ID NO: 231 ctgttttgaagactctcca 5408 5428 SEQ ID NO: 1234 tggagggtagtcataacag 12835 12854 1 5 SEQ ID NO: 232 agtggctgaaacgtgtgca 5441 5460 SEQ ID NO: 1235 tgcagagctttctgccact 14043 14062 1 5 SEQ ID NO: 233 ccaaaatagaagggaatct 5488 5507 SEQ ID NO: 1236 agattcctttctttttcaa 7512 7531 1 5 SEQ ID NO: 234 tgaagagaagattgaattt 5502 5521 SEQ ID NO: 1237 aaattctcttttattttca 5890 5909 1 5 SEQ ID NO: 235 agtggtggcaacaccagca 5544 5583 SEQ ID NO: 1238 tgctagtgaggccaacact 10933 10952 1 5 SEQ ID NO: 236 aaggctccacaagtcatca 5620 5639 SEQ ID NO: 1239 tgatgatatctggaacctt 11204 11223 1 5 SEQ ID NO: 237 gtccgccaggtttctagca 5652 5671 SEQ ID NO: 1240 tgctaagaaccttactgac 7780 7799 1 5 SEQ ID NO: 238 tgatatctggaaccttgga 5667 5686 SEQ ID NO: 1241 ttcactgttcctgaaatca 11746 11765 1 5 SEQ ID NO: 239 gtcaagttgagcaatttct 5865 5885 SEQ ID NO: 1242 agaaaaggcacaccttgac 8036 8057 1 5 SEQ ID NO: 240 atccagatggaaaagggaa 5934 5953 SEQ ID NO: 1243 ttccaatttccctgtggat 10838 10857 1 5 SEQ ID NO: 241 atttgtttgtcaaagaagt 6034 6053 SEQ ID NO: 1244 acttcagagagaaatacat 7599 7618 1 5 SEQ ID NO: 242 ctggaaaatgtcagcctgg 6066 6085 SEQ ID NO: 1245 ccagacttcagttaccagc 8082 8101 1 5 SEQ ID NO: 243 accaggaggttcttcttca 6140 6159 SEQ ID NO: 1246 tgaagtgtgatctcctggt 10924 10943 1 5 SEQ ID NO: 244 aaagaagttctgaaaagat 6192 6211 SEQ ID NO: 1247 attccatcacaaaatcctt 7827 7846 1 5 SEQ ID NO: 245 gctacagcttatggctcca 6217 6236 SEQ ID NO: 1248 tggatctaaatgcagtagc 13174 13193 1 5 SEQ ID NO: 245 atcaatatgatcaatttgt 6295 6314 SEQ ID NO: 1249 caaagaaatcaagattgat 8630 8649 1 5 SEQ ID NO: 247 gaattatctttaaaacatt 6357 6376 SEQ ID NO: 1250 atgtggacaaatataccgg 8294 8313 1 5 SEQ ID NO: 248 cgaggcccgcgctgctggc 6376 6395 SEQ ID NO: 1251 gccagaagtgagatcctcg 11928 11947 1 5 SEQ ID NO: 249 acaactatgaggctgagag 386 6405 SEQ ID NO: 1252 ctctgagcaacaaattttt 13014 13033 1 5 SEQ ID NO: 250 gctgagagttccagtggag 6387 6406 SEQ ID NO: 1253 ctccatggcaaatgtcagg 10052 10071 1 5 SEQ ID NO: 251 tgaagaaaaaccaagaact 6401 3420 SEQ ID NO: 1254 gagtcattgaggttcttca 6984 7003 1 5 SEQ ID NO: 252 cctacttacatcctgaaca 2402 6421 SEQ ID NO: 1255 tgttcataagggaggtagg 6983 7002 1 5 SEQ ID NO: 253 ctacttacatcctgaacat 6405 6425 SEQ ID NO: 1256 atgttcataagggaggtag 8287 8306 1 5 SEQ ID NO: 254 gagacagaagccaagagcc 6414 6433 SEQ ID NO: 1257 gcttggttttgcccagtct 1660 1679 1 5 SEQ ID NO: 255 cactcactttaccgtcaag 6476 6495 SEQ ID NO: 1258 cttgaacaccaaagtcact 6719 6738 1 5 SEQ ID NO: 256 ctgatcagcagcagccagt 6480 6499 SEQ ID NO: 1259 actgggaagtgcttatcag 7094 7113 1 5 SEQ ID NO: 257 actggacgctaagaggaag 6498 6517 SEQ ID NO: 1260 cttccccaaagagaggagt 9488 9507 1 5 SEQ ID NO: 158 agaggaagcatgtggcaga 6693 6712 SEQ ID NO: 1261 tctggcatttactttctct 6806 6825 1 5 SEQ ID NO: 259 tgaagactctccaggaact 6731 6750 SEQ ID NO: 1262 agttgaaggagactattca 6757 6776 1 5 SEQ ID NO: 260 ctctgagcaaaatatccag 6808 6827 SEQ ID NO: 1263 ctggttactgagctgagag 13171 13190 1 5 SEQ ID NO: 261 atgaagcagtcacatctct 6938 6957 SEQ ID NO: 1264 agagctgccagtcttcatt 2458 2477 1 5 SEQ ID NO: 262 ttgccacagctgattgagg 7021 7040 SEQ ID NO: 1265 cctcctacagtggtggcaa 8082 8101 1 5 SEQ ID NO: 263 agctgattgaggtgtccag 7174 7193 SEQ ID NO: 1266 ctggattccacatgcagct 13147 13166 1 5 SEQ ID NO: 264 tgctccactcacatcctcc 7233 7252 SEQ ID NO: 1267 ggaggctttaagttcagca 12545 12564 1 5 SEQ ID NO: 265 tgaaacgtgtgcatgtcaa 7262 7281 SEQ ID NO: 1268 ttgggagagacaagtttca 13155 13174 1 5 SEQ ID NO: 266 gacattgctaattacctga 7269 7288 SEQ ID NO: 1269 tcagaagctaagcaatgtc 9481 9500 1 5 SEQ ID NO: 267 ttcttcttcagactttcct 7281 7300 SEQ ID NO: 1270 aggagagtccaaatttaga 12962 12981 1 5 SEQ ID NO: 268 ccaatatcttgaaactcag 7295 7314 SEQ ID NO: 1271 tctgaattcattcaattgg 7371 7390 1 5 SEQ ID NO: 269 aaagttagtgaaaagaagt 7326 7345 SEQ ID NO: 1272 aactaccctcactgccttt 7677 7696 1 5 SEQ ID NO: 270 aagttagtgaaagaagttc 7403 7422 SEQ ID NO: 1273 gaacctctggcattttact 10731 10750 1 5 SEQ ID NO: 271 aaagaagttctgaaagaat 7540 7559 SEQ ID NO: 1274 attctctggtaactacttt 8412 8431 1 5 SEQ ID NO: 272 tttggctataccaaagatg 7691 7710 SEQ ID NO: 1275 catcttaggcactgacaaa 12001 12020 1 5 SEQ ID NO: 273 tgttgagaagctgattaaa 7950 7969 SEQ ID NO: 1276 tttagccatcggctcaaca 12505 12625 1 5 SEQ ID NO: 274 caggaagggctcaaagaat 7984 8003 SEQ ID NO: 1277 attcctttaacaattcctg 13116 13135 1 5 SEQ ID NO: 275 aggaagggctcaaagaatg 8104 8123 SEQ ID NO: 1278 cattcctttaacaattcct 12352 12371 1 5 SEQ ID NO: 276 gaagggctcaaagaatgac 8148 8167 SEQ ID NO: 1279 gtcagtcttcaggctcttc 8801 8820 1 5 SEQ ID NO: 277 caaagaatgacttttttct 8399 5418 SEQ ID NO: 1280 agaaggatggcattttttg 11009 11028 1 5 SEQ ID NO: 278 catggagaatgcctttgaa 8524 8543 SEQ ID NO: 1281 ttcagagccaaagtccatg 14015 14034 1 5 SEQ ID NO: 279 ggagccaaggctggagtaa 8525 8544 SEQ ID NO: 1282 ttactccaacgccagctcc 9614 9633 1 5 SEQ ID NO: 280 tcattccttccccaaagag 8526 8547 SEQ ID NO: 1283 ctctctggggcatctatgc 14017 14036 1 5 SEQ ID NO: 281 acctatgagctccagagag 8637 8656 SEQ ID NO: 1284 ctctcaagaccacagaagg 11195 11214 1 5 SEQ ID NO: 282 gggcaaaaacgtcttacag 8638 8657 SEQ ID NO: 1285 tctgaaagacaacgtgccc 11194 11213 1 5 SEQ ID NO: 283 accctggaccttcagaaca 8698 8717 SEQ ID NO: 1286 tgttgctaaggttcagggt 10825 10844 1 5 SEQ ID NO: 284 atgggcgacctaagttgtg 8708 8727 SEQ ID NO: 1287 cacaaattagtttcaccat 1261 1300 1 5 SEQ ID NO: 285 gatgaagagaagattgaat 8878 8897 SEQ ID NO: 1288 atccagcttccccacactt 13751 13770 1 5 SEQ ID NO: 286 caatgtgataccaaaaaaa 8883 8902 SEQ ID NO: 1289 tttttggaaatgccattgt 9274 9298 1 5 SEQ ID NO: 287 gtagataccaaaaaaatga 8902 8921 SEQ ID NO: 1290 tcatgtgatgggtctctac 9432 9451 1 5 SEQ ID NO: 288 gcttcagttcatttggact 8916 8935 SEQ ID NO: 1291 agtcaagaaggacttaagc 12406 12427 1 5 SEQ ID NO: 289 tttgtttgtcaaagaagtc 8922 8941 SEQ ID NO: 1292 gacttcagagaaatacaaa 8330 8349 1 5 SEQ ID NO: 290 ttgtttgtcaaagaagtca 5978 8997 SEQ ID NO: 1293 tgacttcagagaaatacaa 11159 11175 1 5 SEQ ID NO: 291 tggcaatgggaaactcgct 9252 9271 SEQ ID NO: 1294 agcgagagtcccctgccat 10219 10238 1 5 SEQ ID NO: 292 aacctctggcatttacttt 9257 9276 SEQ ID NO: 1295 aaaggagatgtcaagggtt 11480 11499 1 5 SEQ ID NO: 293 catttactttctctcatga 9407 9426 SEQ ID NO: 1296 tcatttgaaagaataaatg 9663 9682 1 5 SEQ ID NO: 294 aaagtcagtgccctgctta 9408 9427 SEQ ID NO: 1297 taagaaccttactgacttt 9662 9681 1 5 SEQ ID NO: 295 tcccattttttgagacctt 9417 9436 SEQ ID NO: 1298 aaggacttcaggaatggga 13221 13240 1 5 SEQ ID NO: 296 catcaatattgatcaattt 9418 9437 SEQ ID NO: 1299 aaattaaaaagtcttgatg 13220 13239 1 5 SEQ ID NO: 297 taaagatagttatgattta 9433 9452 SEQ ID NO: 1300 taaaccaaaaacttggtta 8901 8920 1 5 SEQ ID NO: 298 tattgatgaaatcattgaa 9467 9486 SEQ ID NO: 1301 ttcaaagacttaaaaaata 13813 13832 1 5 SEQ ID NO: 299 atgatctacatttgtttat 9557 95786 SEQ ID NO: 1302 ataaagaaattaaagtcat 14013 14032 1 5 SEQ ID NO: 300 agagacacatacagaatat 9704 9723 SEQ ID NO: 1303 atatattgtcagtgcctct 14025 14044 1 5 SEQ ID NO: 301 gacacatacagaatataga 9743 9762 SEQ ID NO: 1304 tctaaattcagttcttgtc 5625 5644 1 5 SEQ ID NO: 302 agcatgtcaaacactttgt 9993 10012 SEQ ID NO: 1305 acaaagtcagtgccctgct 14081 14100 1 5 SEQ ID NO: 303 tttttagaggaaaccaagg 10186 10205 SEQ ID NO: 1306 ccttgtgtacaccaaaaac 4766 4818 1 5 SEQ ID NO: 304 ttttagaggaaaccaaggc 10328 10347 SEQ ID NO: 1307 gcctttgtgtacaccaaaa 2726 2745 1 5 SEQ ID NO: 305 ggaagatagacttcctgaa 10396 10415 SEQ ID NO: 1308 ttcagaaatactgtttttc 8358 8377 1 5 SEQ ID NO: 306 cactgtttctgagtcccag 10397 10416 SEQ ID NO: 1309 ctgggacctaccaagagtg 8357 8376 1 5 SEQ ID NO: 307 cacaaatcctttggctgtg 10428 10447 SEQ ID NO: 1310 cacatttcaaggaattgtg 13250 13269 1 5 SEQ ID NO: 308 ttcctggatacactgttcc 10570 10589 SEQ ID NO: 1311 ggaactgttgactcaggac 12939 12958 1 5 SEQ ID NO: 309 gaaatctcaagctttctct 10655 10674 SEQ ID NO: 1312 agagccaggtcgagctttc 12658 12687 1 5 SEQ ID NO: 310 tttcttcatcttcatctgt 10664 10683 SEQ ID NO: 1313 acagctgaaagagatgaaa 6000 6019 1 5 SEQ ID NO: 311 tctaccgctaaaggagcag 10743 10762 SEQ ID NO: 1314 ctgcacgctttgaggtaga 5279 6298 1 5 SEQ ID NO: 312 ctaccgctaaaggagcagt 10874 10893 SEQ ID NO: 1315 actgcacgctttgaggtcg 11364 11353 1 5 SEQ ID NO: 313 aggggcctcttttcaccaa 11176 11195 SEQ ID NO: 1316 ttggccaggaagtggccct 11847 11836 1 5 SEQ ID NO: 314 tctccatccctgtaaaaag 11477 11496 SEQ ID NO: 1317 ctttttcaccaacggagaa 6757 8775 1 5 SEQ ID NO: 315 gaaaaacaaagcagattat 11605 11624 SEQ ID NO: 1318 ataaactgcaagatttttc 12416 12435 1 5 SEQ ID NO: 316 actcactcattgattttct 11608 11625 SEQ ID NO: 1319 agaaaatcaggatcgtagt 12415 12434 1 5 SEQ ID NO: 317 taaactaatagatgtaatc 11834 11853 SEQ ID NO: 1320 gattaccaccagcagttta 13095 13114 1 5 SEQ ID NO: 318 caaaaacgagcttcggaag 12221 12240 SEQ ID NO: 1321 cttcgtgagaatatttgaa 13623 13642 1 5 SEQ ID NO: 319 tggaataatgctcagtgtt 12930 12949 SEQ ID NO: 1322 aacacttaacttgaattca 13818 13837 1 5 SEQ ID NO: 320 gatttgaaatccaaagaag 13026 13045 SEQ ID NO: 1323 cttcagagaaatacaaatc 8005 8025 1 5 SEQ ID NO: 321 atttgaaatccaaagaagt 13089 13108 SEQ ID NO: 1324 acttcagagaaatacaaat 10471 10490 1 5 SEQ ID NO: 322 atcaacagccgcttctttg 13210 13229 SEQ ID NO: 1325 caaagaagtcaagattgat 13175 13194 1 5 SEQ ID NO: 323 tgttttgaagactctccag 13429 13448 SEQ ID NO: 1326 ctggaaagttaaaacaaac 12924 12943 1 5 SEQ ID NO: 324 cccttctgatagatgtggt 13704 13723 SEQ ID NO: 1327 accaaagctggcaccaggg 7457 7475 1 5 SEQ ID NO: 325 tgagcaagtgaagaacttt 13711 13730 SEQ ID NO: 1328 aaagccattcagtctctca 7231 7250 1 5 SEQ ID NO: 326 atttgaaatccaaagaagt 13874 13893 SEQ ID NO: 1329 actttctaaacttgaaatt 8372 8391 1 5 SEQ ID NO: 327 atccaaagaagtcccggaa 14003 14022 SEQ ID NO: 1330 tccggggaaacctgggatt 3005 3024 1 5 SEQ ID NO: 328 agagcctacctccgcatct 14012 14031 SEQ ID NO: 1331 agatggtacgttagcctct 9558 9577 1 5 SEQ ID NO: 329 aatgcctttgaactcccca 1619 1638 SEQ ID NO: 1332 tgggaactacaattcattt 8633 8662 3 4 SEQ ID NO: 330 gaagtccaaattccggatt 1948 1967 SEQ ID NO: 1333 aatcttcaatttattcttc 12555 12575 3 4 SEQ ID NO: 331 tgcaagcagaagccagaag 5427 5446 SEQ ID NO: 1334 cttcaggttccatcgtgca 11401 11420 3 4 SEQ ID NO: 332 gaagagaagattgaatttg 6480 8499 SEQ ID NO: 1335 caaaacctactgtgcttcc 7421 7440 3 4 SEQ ID NO: 333 atgctaaaggcacatatgg 18 37 SEQ ID NO: 1336 ccatatgaaagtcaagcat 6031 6050 2 4 SEQ ID NO: 334 tccctcacctccacctctg 245 264 SEQ ID NO: 1337 cagattctcagatgaggga 13176 13195 2 4 SEQ ID NO: 335 atttacagctctgacaagt 308 327 SEQ ID NO: 1338 acttttctaaacttgaaat 13407 13426 2 4 SEQ ID NO: 336 aggagcctaccaaaataat 475 494 SEQ ID NO: 1339 attatgttgaaaacagtct 5881 5900 2 4 SEQ ID NO: 337 aaagctgaagcacatcaat 547 566 SEQ ID NO: 1340 attgttgctcatctccttt 10490 10509 2 4 SEQ ID NO: 338 ctgctggaaacaacgagaa 1087 1106 SEQ ID NO: 1341 ttctgattaccaccagcag 13183 13202 2 4 SEQ ID NO: 339 ttgaaggaattcttgaaaa 1202 1221 SEQ ID NO: 1342 ttttaaaagaaatcttcaa 9229 9248 2 4 SEQ ID NO: 340 gaagtaaaagaaaattttg 1203 1222 SEQ ID NO: 1343 caaaacctactgtctcttc 9226 9247 2 4 SEQ ID NO: 341 tgaagaagatggcaaattt 1223 1242 SEQ ID NO: 1344 aaatgtcagctcttgttca 5208 5227 2 4 SEQ ID NO: 342 aggatctgagttatttttc 1620 1639 SEQ ID NO: 1345 gcaagtcagcccagttcct 5907 5926 2 4 SEQ ID NO: 343 gtgcccttctcggttgctg 1941 1960 SEQ ID NO: 1346 cagccattgacatgagcac 10623 10642 2 4 SEQ ID NO: 344 ggcgcggcctgcgctgctg 2238 2257 SEQ ID NO: 1347 cagctccacagactccgcc 9722 9741 2 4 SEQ ID NO: 345 ctgcgctgctgctgctgct 2239 2258 SEQ ID NO: 1348 agcagaaggtgcgaagcag 9721 9740 2 4 SEQ ID NO: 346 gctggtggcgggcgccagg 3395 3414 SEQ ID NO: 1349 cctggattccacatgcagc 10406 10425 2 4 SEQ ID NO: 347 aagaggaaatgctggaaaa 3620 3639 SEQ ID NO: 1350 tttttcttcactacatctt 10894 10913 2 4 SEQ ID NO: 348 ctggaaaatgtcagcctgg 3636 3655 SEQ ID NO: 1351 ccagacttccacatcccag 11807 11826 2 4 SEQ ID NO: 349 tggagtccctgggactgct 4399 4416 SEQ ID NO: 1352 agcatgcctagtttctcca 7369 7388 2 4 SEQ ID NO: 350 ggagtccctgggactgctg 4404 4423 SEQ ID NO: 1353 cagcatgcctagtttctcc 7154 7173 2 4 SEQ ID NO: 351 tgggactgctgattcaaga 5075 5094 SEQ ID NO: 1354 tcttccatcacttgaccca 11376 11395 2 4 SEQ ID NO: 352 ctgctgattcaagaagtgc 5317 5336 SEQ ID NO: 1355 gcacaccttgacattgcag 7374 7393 2 4 SEQ ID NO: 353 tgccaccaggatcaactgc 6066 6085 SEQ ID NO: 1356 gcaggctgaactggtggca 13558 13887 2 4 SEQ ID NO: 354 gccaccaggatcaactgca 6080 6099 SEQ ID NO: 1357 tgcaggctgaactggtggc 10679 10698 2 4 SEQ ID NO: 355 tgcaaggttgagcgtggag 6413 6432 SEQ ID NO: 1358 cctccatcctctgatctga 11478 11497 2 4 SEQ ID NO: 356 caaggttgagctggaggtt 7051 7070 SEQ ID NO: 1359 aacccctacatgaagcttg 9373 9392 2 4 SEQ ID NO: 357 ctctgcagcttcatcctga 7219 7238 SEQ ID NO: 1360 tcaggaagcttctcaagag 13438 13457 2 4 SEQ ID NO: 358 cagcttcatcctgaagaac 7921 7940 SEQ ID NO: 1361 ggtcttgagttaaatgctg 12304 12323 2 4 SEQ ID NO: 359 gcttcatcctgaagaccag 8779 8798 SEQ ID NO: 1362 ctggacgctaagaggaagc 12525 12544 2 4 SEQ ID NO: 360 tcatcctgaagaccagcca 10159 10178 SEQ ID NO: 1363 tggcatggcattatgatga 13183 13202 2 4 SEQ ID NO: 361 gaaaaccaagaactctgag 12890 12909 SEQ ID NO: 1364 ctcaaccttaatgattttc 13632 13651 2 4 SEQ ID NO: 362 agaactctgaggagtttgc 13672 13691 SEQ ID NO: 1365 gcaagctatatcagtattc 13805 13824 2 4 SEQ ID NO: 363 tctgaggagtttgctgcag 11 30 SEQ ID NO: 1366 ctgcagggatcccccagat 76 95 1 4 SEQ ID NO: 364 tttgctgcagccatgtcca 12 31 SEQ ID NO: 1367 tggaagtgtcagtggcaaa 75 94 1 4 SEQ ID NO: 365 caagaggggcatcatttct 14 33 SEQ ID NO: 1368 agaataaatgacgttcttg 11549 11568 1 4 SEQ ID NO: 366 tcactttaccgtcaagacg 25 44 SEQ ID NO: 1369 cgtctacactatcatgtga 2160 2179 1 4 SEQ ID NO: 367 tttaccgtcaagacgagga 82 101 SEQ ID NO: 1370 tccttgacatgttgataaa 368 367 1 4 SEQ ID NO: 368 cactggacgctaagaggaa 143 162 SEQ ID NO: 1371 ttccagaaagcagccagtg 4244 4263 1 4 SEQ ID NO: 369 aggaagcatgtggcagaag 169 188 SEQ ID NO: 1372 cttcatacacattaatcct 11178 11197 1 4 SEQ ID NO: 370 caaggagcaacacctcttc 219 238 SEQ ID NO: 1373 gaagtagtactgcatgttg 380 399 1 4 SEQ ID NO: 371 acagactttgaaacttgaa 283 302 SEQ ID NO: 1374 ttcaattcttcaatgctgt 3383 3402 1 4 SEQ ID NO: 372 tgatgaagcagtcacatct 291 310 SEQ ID NO: 1375 agatttgaggattccatca 2675 2694 1 4 SEQ ID NO: 373 agcagtcacatctctcttg 348 365 SEQ ID NO: 1376 caaggagaaactgactcgt 4728 4747 1 4 SEQ ID NO: 374 ccagccccatcactttaca 350 369 SEQ ID NO: 1377 tgtagtctcctggtgctgg 9163 9182 1 4 SEQ ID NO: 375 ctccactccatcctccagg 370 389 SEQ ID NO: 1378 ctggagcttagtaatggag 3261 3280 1 4 SEQ ID NO: 376 catgccaacccccttctga 394 413 SEQ ID NO: 1379 tcagatgagggaacacatg 5794 5813 1 4 SEQ ID NO: 377 gagagatcttcaacatggc 464 483 SEQ ID NO: 1380 gccaccctggaactctctc 6340 6359 1 4 SEQ ID NO: 378 tcaacatggcgagggatca 492 511 SEQ ID NO: 1381 tgatcccacctctcattga 12716 12735 1 4 SEQ ID NO: 379 ccaccttgtatgcgctgag 535 554 SEQ ID NO: 1382 ctcagggatctgaaggtgg 2219 2238 1 4 SEQ ID NO: 380 gtcaacaactatcataaga 575 594 SEQ ID NO: 1383 tcttgagttaaatgctgac 2277 2296 1 4 SEQ ID NO: 381 tggacattgctaattacct 601 620 SEQ ID NO: 1384 aggtatattcgaaagtcca 13988 140007 1 4 SEQ ID NO: 382 ggacattgctaattacctg 622 641 SEQ ID NO: 1385 caggtatattcgaaagtcc 14042 14091 1 4 SEQ ID NO: 383 ttctgcgggtcattggaaa 630 649 SEQ ID NO: 1386 tttcacatgccaaggagaa 11042 11061 1 4 SEQ ID NO: 384 ccagaactcaagtcttcaa 644 663 SEQ ID NO: 1387 ttgaagtgtatgtctcctg 11356 11375 1 4 SEQ ID NO: 385 agtcttcaatcctgaaatg 670 689 SEQ ID NO: 1388 catttctgattggtggact 6817 6836 1 4 SEQ ID NO: 386 tgagcaagtgaagaacttt 693 712 SEQ ID NO: 1389 aaagtgccacttttactca 3005 3024 1 4 SEQ ID NO: 387 agcaagtgaagaactttgt 700 719 SEQ ID NO: 1390 acaaagtcagtgccctgct 3480 3499 1 4 SEQ ID NO: 388 tctgaaagaatctcaactt 701 720 SEQ ID NO: 1391 aagtccataatggttcaga 13826 13845 1 4 SEQ ID NO: 389 actgtcatggacttcagaa 706 725 SEQ ID NO: 1392 ttctgaatatattgtcagt 1887 1906 1 4 SEQ ID NO: 390 acttgacccagcctcagcc 729 748 SEQ ID NO: 1393 ggctcaccctgagagaagt 2930 2949 1 4 SEQ ID NO: 391 tccaaataactaccttcct 744 763 SEQ ID NO: 1394 aggaagatatgaagatgga 5210 5229 1 4 SEQ ID NO: 392 actaccctcactgcctttg 745 764 SEQ ID NO: 1395 caaatttgtggagggtagt 5209 5226 1 4 SEQ ID NO: 393 ttggatttgcttcagctga 776 795 SEQ ID NO: 1396 tcagtataagtacaaccaa 8144 8163 1 4 SEQ ID NO: 394 ttggaagctctttttggga 786 805 SEQ ID NO: 1397 tcccgattcacgcttccaa 10337 10356 1 4 SEQ ID NO: 395 ggaagctctttttgggaag 811 830 SEQ ID NO: 1398 cttcagaaagctaccttcc 12445 12464 1 4 SEQ ID NO: 396 tttttcccagacagtgtca 812 831 SEQ ID NO: 1399 tgaccttctctaagcaaaa 12444 12463 1 4 SEQ ID NO: 397 agacagtgtcaacaaagct 884 903 SEQ ID NO: 1400 agcttggttttgccagtct 3805 3824 1 4 SEQ ID NO: 398 ctttggctataccaaagat 885 904 SEQ ID NO: 1401 atctcgtgtctaggaaaag 3804 3823 1 4 SEQ ID NO: 399 caaagatgataaacatgag 908 927 SEQ ID NO: 1402 ctcaaggataacgtgtttg 9453 9472 1 4 SEQ ID NO: 400 gatatggtaaatggaataa 916 935 SEQ ID NO: 1403 ttatcttataattatatca 13648 13667 1 4 SEQ ID NO: 401 ggaataatgctcagtgttg 989 1008 SEQ ID NO: 1404 caaacacttacttgaattc 1661 1580 1 4 SEQ ID NO: 402 tttgaaatccaaagaagtc 990 1009 SEQ ID NO: 1405 gacttcagagaaatacaaa 1660 1579 1 4 SEQ ID NO: 403 gatcccccagatgattgga 994 1013 SEQ ID NO: 1406 tccaatttccctgtggatc 9667 9686 1 4 SEQ ID NO: 404 cagatattggagaggtcaa 1023 1042 SEQ ID NO: 1407 tgaccacacaaaacagtct 2069 2068 1 4 SEQ ID NO: 405 agaatgacttttttctctc 1082 1101 SEQ ID NO: 1408 tgaagtccggattcattct 5487 5505 1 4 SEQ ID NO: 406 gaactccccactggagctg 1086 1105 SEQ ID NO: 1409 cagctcaaccgtacagttc 13184 13203 1 4 SEQ ID NO: 407 atatcttcatgggagtcaa 1102 1121 SEQ ID NO: 1410 tgacttcagtgcagaatat 14006 14025 1 4 SEQ ID NO: 408 gtcatgctccccggaggca 1104 1123 SEQ ID NO: 1411 tggccccgtttaccatgac 4564 4583 1 4 SEQ ID NO: 409 gctgaagtttatcattcct 1109 1128 SEQ ID NO: 1412 aggaggctttaagttcagc 2570 2589 1 4 SEQ ID NO: 410 attccttccccaaagagac 1124 1143 SEQ ID NO: 1413 gtctcttcctccatggaat 12201 12220 1 4 SEQ ID NO: 411 ctcattgagaacaggcagt 1154 1173 SEQ ID NO: 1414 actgactgcacgctttgag 12265 12284 1 4 SEQ ID NO: 412 ttgagcagtattctgtcag 1166 1185 SEQ ID NO: 1415 cttagagaagtgtcttcaa 10329 10348 1 4 SEQ ID NO: 413 accttgtccagtgaagtcc 1180 1199 SEQ ID NO: 1416 ggacggtactgtcccaggt 12572 12591 1 4 SEQ ID NO: 414 ccagtgaagtccaaattcc 1196 1215 SEQ ID NO: 1417 ggaaggcagagtttactgg 8858 8877 1 4 SEQ ID NO: 415 acattcagaacaagaaaat 1202 1221 SEQ ID NO: 1418 atttcctaaagctggatgc 2161 2180 1 4 SEQ ID NO: 416 gaaaaatcaagggtgttat 1203 1222 SEQ ID NO: 1419 ataaactgcaagatttttc 2160 2179 1 4 SEQ ID NO: 417 aaatcaagggtgttatttc 1210 1229 SEQ ID NO: 1420 gaaacaatgcattagattt 13955 13974 1 4 SEQ ID NO: 418 tggcattatgatgaagaga 1211 1230 SEQ ID NO: 1421 tctcccgtgtataatgcca 11240 11259 1 4 SEQ ID NO: 419 aagagaagattgaatttga 1240 1259 SEQ ID NO: 1422 tcaaaacctactgtctcct 8140 8159 1 4 SEQ ID NO: 420 aaatgacttccaatttccc 1324 1343 SEQ ID NO: 1423 gggaactacaatttcattt 10816 10835 1 4 SEQ ID NO: 421 atgacttccaatttccctg 1341 1390 SEQ ID NO: 1424 caggctgattacgagtcat 3431 3460 1 4 SEQ ID NO: 422 acttccaatttccctgtgg 1432 1451 SEQ ID NO: 1425 ccacgaaaaatatggaagt 5578 5597 1 4 SEQ ID NO: 423 agttgcaatgagctcatgg 1472 1491 SEQ ID NO: 1426 ccatcagttcagataaact 12347 12366 1 4 SEQ ID NO: 424 tttgcaagaccacctcaat 1508 1527 SEQ ID NO: 1427 attgacctgtccattcaaa 9930 9949 1 4 SEQ ID NO: 425 gaaggagttcaacctccag 1538 1557 SEQ ID NO: 1428 ctggaattgtcattccttc 10909 10926 1 4 SEQ ID NO: 426 acttccacatcccagaaaa 1540 1559 SEQ ID NO: 1429 ttttaacaaaaagtggaag 5999 5018 1 4 SEQ ID NO: 427 ctcttcttaaaaagcgatg 1552 1571 SEQ ID NO: 1430 catcactgccaaaggagag 8110 5129 1 4 SEQ ID NO: 428 aaaagcgatggccgggtcc 1602 1621 SEQ ID NO: 1431 tgactcactcattgatttt 9016 9035 1 4 SEQ ID NO: 429 ttcctttgccttttggtgg 1610 1629 SEQ ID NO: 1432 ccacaaaacaatgaaggga 13712 13738 1 4 SEQ ID NO: 430 caagtctgtgggattccat 1621 1640 SEQ ID NO: 1433 atgggaaaaaacaggcttg 1925 1944 1 4 SEQ ID NO: 431 aagtccctacttttaccat 1695 1714 SEQ ID NO: 1434 atgggaagtataagaactt 2477 2496 1 4 SEQ ID NO: 432 tgcctctcctgggtgttct 1730 1749 SEQ ID NO: 1435 agaaaaacaaacaaacgga 5511 5630 1 4 SEQ ID NO: 433 accagcacagaccatttca 1736 1755 SEQ ID NO: 1436 tgaagtgtagtctcctggt 4431 4450 1 4 SEQ ID NO: 434 ccagcacagaccatttcag 1751 1770 SEQ ID NO: 1437 ctgaaatacaatgctctgg 6885 6904 1 4 SEQ ID NO: 435 actatcatgtgatgggtct 1773 1792 SEQ ID NO: 1438 agacacctgattttatagt 9944 9963 1 4 SEQ ID NO: 436 accacagatgtctgcttca 1788 1807 SEQ ID NO: 1439 tgaaggctgactctgtggt 12037 12056 1 4 SEQ ID NO: 437 ccacagagtgtctgcttcg 1882 1901 SEQ ID NO: 1440 ctgagcaacaatttgtgga 10729 10748 1 4 SEQ ID NO: 438 tttggactccaaaaagaaa 1902 1921 SEQ ID NO: 1441 tttctctcatgattacaaa 13992 14011 1 4 SEQ ID NO: 439 tcaaagaagtcaagattga 1905 1924 SEQ ID NO: 1442 tcaaggataactgtttgag 13370 13389 1 4 SEQ ID NO: 440 atgagaactacgagctgac 1916 1935 SEQ ID NO: 1443 gtcagatattgttgctcat 7265 7284 1 4 SEQ ID NO: 441 ttaaactctgacaccaatg 1921 1940 SEQ ID NO: 1444 cattcattgaagatgttaa 13817 13836 1 4 SEQ ID NO: 442 gaagtataagaactttgcc 1922 1941 SEQ ID NO: 1445 ggcaaatttgaaggacttc 13816 13835 1 4 SEQ ID NO: 443 aagtataagaaactttgca 1927 1943 SEQ ID NO: 1446 tggcaaatttgaaggactt 8317 8338 1 4 SEQ ID NO: 444 ttcttcagcctgctttctg 1930 1949 SEQ ID NO: 1447 cagaatccagatacaagaa 10177 10196 1 4 SEQ ID NO: 445 ctggatcactaaattccca 1931 1950 SEQ ID NO: 1448 tgggtctttccagagcccg 10176 10195 1 4 SEQ ID NO: 446 aaattaatagtggtgctca 1935 1954 SEQ ID NO: 1449 tgagaagccccaagaattt 4965 4984 1 4 SEQ ID NO: 447 ggccattccagaagggaag 1936 1955 SEQ ID NO: 1450 cttccgttctgtaatggcc 4964 4983 1 4 SEQ ID NO: 448 tgccatctcgagagttcca 1942 1961 SEQ ID NO: 1451 tggaactctctccatggca 10622 10641 1 4 SEQ ID NO: 449 catgtcaaacactttgtta 1682 2001 SEQ ID NO: 1452 taacaaattccttgacatg 13937 13956 1 4 SEQ ID NO: 450 tttgttataaatcttattg 1999 2018 SEQ ID NO: 1453 caataagatcaatagcaaa 9493 9512 1 4 SEQ ID NO: 451 tctggaaaaagggtcatga 2044 2063 SEQ ID NO: 1454 tccatgtcccatttacaga 8433 8452 1 4 SEQ ID NO: 452 cagctcttgttcaggtcca 2057 2076 SEQ ID NO: 1455 tggacctgcaccaaagctg 7812 7831 1 4 SEQ ID NO: 453 ggaggttccccagctctgc 2060 2079 SEQ ID NO: 1456 gcagccctgggaaaactcc 7814 7833 1 4 SEQ ID NO: 454 ctgttttgaagactctcca 2063 2102 SEQ ID NO: 1457 tggagggtagtcataacag 11813 11832 1 4 SEQ ID NO: 455 agtggctgaaacgtgtgca 2084 213 SEQ ID NO: 1458 tgcagagctttctgccact 14011 14030 1 4 SEQ ID NO: 456 ccaaaatagaagggaatct 2109 2126 SEQ ID NO: 1459 agattcctttctttttcaa 3605 3625 1 4 SEQ ID NO: 457 tgaagagaagattgaattt 2113 2132 SEQ ID NO: 1460 aaattctcttttattttca 5686 5705 1 4 SEQ ID NO: 458 agtggtggcaacaccagca 2114 2133 SEQ ID NO: 1461 tgctagtgaggccaacact 9482 9501 1 4 SEQ ID NO: 459 aaggctccacaagtcatca 2175 2194 SEQ ID NO: 1462 tgatgatatctggaacctt 11701 11720 1 4 SEQ ID NO: 460 gtccgccaggtttctagca 2198 2217 SEQ ID NO: 1463 tgctaagaaccttactgac 11060 11079 1 4 SEQ ID NO: 461 tgatatctggaaccttgga 2245 2264 SEQ ID NO: 1464 ttcactgttcctgaaatca 6134 6153 1 4 SEQ ID NO: 462 gtcaagttgagcaatttct 2249 2268 SEQ ID NO: 1465 agaaaaggcacaccttgac 9849 9868 1 4 SEQ ID NO: 463 atccagatggaaaagggaa 2250 2269 SEQ ID NO: 1466 ttccaatttccctgtggat 4351 4370 1 4 SEQ ID NO: 464 atttgtttgtcaaagaagt 2290 2309 SEQ ID NO: 1467 acttcagagagaaatacat 3325 3344 1 4 SEQ ID NO: 465 ctggaaaatgtcagcctgg 2291 2310 SEQ ID NO: 1468 ccagacttcagttaccagc 8823 6642 1 4 SEQ ID NO: 466 accaggaggttcttcttca 2343 2362 SEQ ID NO: 1469 tgaagtgtgatctcctggt 6788 6807 1 4 SEQ ID NO: 467 aaagaagttctgaaaagat 2387 2406 SEQ ID NO: 1470 attccatcacaaaatcctt 5279 5298 1 4 SEQ ID NO: 468 gctacagcttatggctcca 2397 2416 SEQ ID NO: 1471 tggatctaaatgcagtagc 7606 7625 1 4 SEQ ID NO: 469 atcaatatgatcaatttgt 2510 2529 SEQ ID NO: 1472 caaagaaatcaagattgat 7975 7994 1 4 SEQ ID NO: 470 gaattatctttaaaacatt 2532 2551 SEQ ID NO: 1473 atgtggacaaatataccgg 9075 9094 1 4 SEQ ID NO: 471 cgaggcccgcgctgctggc 2586 2604 SEQ ID NO: 1474 gccagaagtgagatcctcg 10374 10393 1 4 SEQ ID NO: 472 acaactatgaggctgagag 2588 2607 SEQ ID NO: 1475 ctctgagcaacaaattttt 3607 3626 1 4 SEQ ID NO: 473 gctgagagttccagtggag 2595 2614 SEQ ID NO: 1476 ctccatggcaaatgtcagg 9437 9458 1 4 SEQ ID NO: 474 tgaagaaaaaccaagaact 2601 2620 SEQ ID NO: 1477 gagtcattgaggttcttca 6599 6618 1 4 SEQ ID NO: 475 cctacttacatcctgaaca 2602 2621 SEQ ID NO: 1478 tgttcataagggaggtagg 13108 13127 1 4 SEQ ID NO: 476 ctacttacatcctgaacat 2616 2635 SEQ ID NO: 1479 atgttcataagggaggtag 9834 9853 1 4 SEQ ID NO: 477 gagacagaagccaagagcc 2617 2636 SEQ ID NO: 1480 gcttggttttgcccagtct 9833 9852 1 4 SEQ ID NO: 478 cactcactttaccgtcaag 2618 2637 SEQ ID NO: 1481 cttgaacaccaaagtcact 9832 9651 1 4 SEQ ID NO: 479 ctgatcagcagcagccagt 2627 2646 SEQ ID NO: 1482 actgggaagtgcttatcag 9336 9355 1 4 SEQ ID NO: 480 actggacgctaagaggaag 2628 2647 SEQ ID NO: 1483 cttccccaaagagaggagt 9335 9354 1 4 SEQ ID NO: 481 agaggaagcatgtggcaga 2644 2663 SEQ ID NO: 1484 tctggcatttactttctct 6591 6610 1 4 SEQ ID NO: 482 tgaagactctccaggaact 2645 2664 SEQ ID NO: 1485 agttgaaggagactattca 6590 6609 1 4 SEQ ID NO: 483 ctctgagcaaaatatccag 2650 2689 SEQ ID NO: 1486 ctggttactgagctgagag 13996 14015 1 4 SEQ ID NO: 484 atgaagcagtcacatctct 2695 2714 SEQ ID NO: 1487 agagctgccagtcttcatt 7592 7611 1 4 SEQ ID NO: 485 ttgccacagctgattgagg 2720 2739 SEQ ID NO: 1488 cctcctacagtggtggcaa 9220 9239 1 4 SEQ ID NO: 486 agctgattgaggtgtccag 2750 2789 SEQ ID NO: 1489 ctggattccacatgcagct 8530 8549 1 4 SEQ ID NO: 487 tgctccactcacatcctcc 2758 2777 SEQ ID NO: 1490 ggaggctttaagttcagca 9513 9532 1 4 SEQ ID NO: 488 tgaaacgtgtgcatgtcaa 2811 2830 SEQ ID NO: 1491 ttgggagagacaagtttca 2669 2888 1 4 SEQ ID NO: 489 gacattgctaattacctga 2825 2844 SEQ ID NO: 1492 tcagaagctaagcaatgtc 5227 5246 1 4 SEQ ID NO: 490 ttcttcttcagactttcct 2832 2851 SEQ ID NO: 1493 aggagagtccaaatttaga 5132 6151 1 4 SEQ ID NO: 491 ccaatatcttgaaactcag 2858 2877 SEQ ID NO: 1494 tctgaattcattcaattgg 12112 12131 1 4 SEQ ID NO: 492 aaagttagtgaaaagaagt 2664 2883 SEQ ID NO: 1495 aactaccctcactgccttt 5455 5474 1 4 SEQ ID NO: 493 aagttagtgaaagaagttc 3106 3125 SEQ ID NO: 1496 gaacctctggcattttact 12718 12737 1 4 SEQ ID NO: 494 aaagaagttctgaaagaat 3200 3219 SEQ ID NO: 1497 attctctggtaactacttt 8385 8404 1 4 SEQ ID NO: 495 tttggctataccaaagatg 3244 3263 SEQ ID NO: 1498 catcttaggcactgacaaa 3809 3828 1 4 SEQ ID NO: 496 tgttgagaagctgattaaa 3286 3308 SEQ ID NO: 1499 tttagccatcggctcaaca 6349 6968 1 4 SEQ ID NO: 497 caggaagggctcaaagaat 3305 3324 SEQ ID NO: 1500 attcctttaacaattcctg 8930 8949 1 4 SEQ ID NO: 498 aggaagggctcaaagaatg 3307 3326 SEQ ID NO: 1501 cattcctttaacaattcct 13199 13216 1 4 SEQ ID NO: 499 gaagggctcaaagaatgac 3329 3348 SEQ ID NO: 1502 gtcagtcttcaggctcttc 4203 4222 1 4 SEQ ID NO: 500 caaagaatgacttttttct 3337 3356 SEQ ID NO: 1503 agaaggatggcattttttg 7585 7604 1 4 SEQ ID NO: 501 catggagaatgcctttgaa 3384 3403 SEQ ID NO: 1504 ttcagagccaaagtccatg 12431 12450 1 4 SEQ ID NO: 502 ggagccaaggctggagtaa 3395 3414 SEQ ID NO: 1505 ttactccaacgccagctcc 8096 8115 1 4 SEQ ID NO: 503 tcattccttccccaaagag 3414 3433 SEQ ID NO: 1506 ctctctggggcatctatgc 10916 10935 1 4 SEQ ID NO: 504 acctatgagctccagagag 3478 3497 SEQ ID NO: 1507 ctctcaagaccacagaagg 13672 13891 1 4 SEQ ID NO: 505 gggcaaaaacgtcttacag 3493 3512 SEQ ID NO: 1508 tctgaaagacaacgtgccc 5400 5419 1 4 SEQ ID NO: 506 accctggaccttcagaaca 3494 3513 SEQ ID NO: 1509 tgttgctaaggttcagggt 5264 5283 1 4 SEQ ID NO: 507 atgggcgacctaagttgtg 3495 3514 SEQ ID NO: 1510 cacaaattagtttcaccat 5263 5282 1 4 SEQ ID NO: 508 gatgaagagaagattgaat 3546 3565 SEQ ID NO: 1511 atccagcttccccacactt 4559 4578 1 4 SEQ ID NO: 509 caatgtgataccaaaaaaa 3589 3588 SEQ ID NO: 1512 tttttggaaatgccattgt 7601 760 1 4 SEQ ID NO: 510 gtagataccaaaaaaatga 3573 3592 SEQ ID NO: 1513 tcatgtgatgggtctctac 5889 5908 1 4 SEQ ID NO: 511 gcttcagttcatttggact 3592 3611 SEQ ID NO: 1514 agtcaagaaggacttaagc 8869 8888 1 4 SEQ ID NO: 512 tttgtttgtcaaagaagtc 3695 3614 SEQ ID NO: 1515 gacttcagagaaatacaaa 9353 9372 1 4 SEQ ID NO: 513 ttgtttgtcaaagaagtca 3603 3622 SEQ ID NO: 1516 tgacttcagagaaatacaa 8063 8082 1 4 SEQ ID NO: 514 tggcaatgggaaactcgct 3617 3636 SEQ ID NO: 1517 agcgagagtcccctgccat 7863 7682 1 4 SEQ ID NO: 515 aacctctggcatttacttt 3621 3640 SEQ ID NO: 1518 aaaggagatgtcaagggtt 5279 5298 1 4 SEQ ID NO: 516 catttactttctctcatga 3624 3643 SEQ ID NO: 1519 tcatttgaaagaataaatg 8270 8289 1 4 SEQ ID NO: 517 aaagtcagtgccctgctta 3638 3655 SEQ ID NO: 1520 taagaaccttactgacttt 11228 11247 1 4 SEQ ID NO: 518 tcccattttttgagacctt 3850 3559 SEQ ID NO: 1521 aaggacttcaggaatggga 5583 5602 1 4 SEQ ID NO: 519 catcaatattgatcaattt 3668 3667 SEQ ID NO: 1522 aaattaaaaagtcttgatg 8358 8377 1 4 SEQ ID NO: 520 taaagatagttatgattta 3669 3688 SEQ ID NO: 1523 taaaccaaaaacttggtta 8357 8376 1 4 SEQ ID NO: 521 tattgatgaaatcattgaa 3670 3689 SEQ ID NO: 1524 ttcaaagacttaaaaaata 8398 8417 1 4 SEQ ID NO: 522 atgatctacatttgtttat 3752 3771 SEQ ID NO: 1525 ataaagaaattaaagtcat 4082 4101 1 4 SEQ ID NO: 523 agagacacatacagaatat 3785 3814 SEQ ID NO: 1526 atatattgtcagtgcctct 13178 13197 1 4 SEQ ID NO: 524 gacacatacagaatataga 3891 3910 SEQ ID NO: 1527 tctaaattcagttcttgtc 8134 8153 1 4 SEQ ID NO: 525 agcatgtcaaacactttgt 3907 3926 SEQ ID NO: 1528 acaaagtcagtgccctgct 8895 8914 1 4 SEQ ID NO: 526 tttttagaggaaaccaagg 3986 4005 SEQ ID NO: 1529 ccttgtgtacaccaaaaac 9533 9552 1 4 SEQ ID NO: 527 ttttagaggaaaccaaggc 3992 4011 SEQ ID NO: 1530 gcctttgtgtacaccaaaa 13686 13076 1 4 SEQ ID NO: 528 ggaagatagacttcctgaa 4007 4026 SEQ ID NO: 1531 ttcagaaatactgtttttc 6351 6370 1 4 SEQ ID NO: 529 cactgtttctgagtcccag 4028 4047 SEQ ID NO: 1532 ctgggacctaccaagagtg 7586 7605 1 4 SEQ ID NO: 530 cacaaatcctttggctgtg 4037 4056 SEQ ID NO: 1533 cacatttcaaggaattgtg 10025 10044 1 4 SEQ ID NO: 531 ttcctggatacactgttcc 4084 4103 SEQ ID NO: 1534 ggaactgttgactcaggac 9727 9746 1 4 SEQ ID NO: 532 gaaatctcaagctttctct 4096 4115 SEQ ID NO: 1535 agagccaggtcgagctttc 8548 8567 1 4 SEQ ID NO: 533 tttcttcatcttcatctgt 4104 4123 SEQ ID NO: 1536 acagctgaaagagatgaaa 8207 8225 1 4 SEQ ID NO: 534 tctaccgctaaaggagcag 4116 4137 SEQ ID NO: 1537 ctgcacgctttgaggtaga 6076 6097 1 4 SEQ ID NO: 535 ctaccgctaaaggagcagt 4125 4144 SEQ ID NO: 1538 actgcacgctttgaggtcg 8101 8120 1 4 SEQ ID NO: 536 aggggcctcttttcaccaa 4133 4152 SEQ ID NO: 1539 ttggccaggaagtggccct 12001 12020 1 4 SEQ ID NO: 537 tctccatccctgtaaaaag 4276 4295 SEQ ID NO: 1540 ctttttcaccaacggagaa 9016 9035 1 4 SEQ ID NO: 538 gaaaaacaaagcagattat 4309 4326 SEQ ID NO: 1541 ataaactgcaagatttttc 9495 9514 1 4 SEQ ID NO: 539 actcactcattgattttct 4330 4349 SEQ ID NO: 1542 agaaaatcaggatcgtagt 11027 11046 1 4 SEQ ID NO: 540 taaactaatagatgtaatc 4370 4389 SEQ ID NO: 1543 gattaccaccagcagttta 6557 6576 1 4 SEQ ID NO: 541 caaaaacgagcttcggaag 4372 4391 SEQ ID NO: 1544 cttcgtgagaatatttgaa 12125 12144 1 4 SEQ ID NO: 542 tggaataatgctcagtgtt 4399 4418 SEQ ID NO: 1545 aacacttaacttgaattca 7300 7319 1 4 SEQ ID NO: 543 gatttgaaatccaaagaag 4491 4510 SEQ ID NO: 1546 cttcagagaaatacaaatc 8325 8345 1 4 SEQ ID NO: 544 atttgaaatccaaagaagt 4636 4655 SEQ ID NO: 1547 acttcagagaaatacaaat 6976 6995 1 4 SEQ ID NO: 545 atcaacagccgcttctttg 4637 4656 SEQ ID NO: 1548 caaagaagtcaagattgat 6975 6994 1 4 SEQ ID NO: 546 tgttttgaagactctccag 4638 4667 SEQ ID NO: 1549 ctggaaagttaaaacaaac 6974 6993 1 4 SEQ ID NO: 547 cccttctgatagatgtggt 4642 4681 SEQ ID NO: 1550 accaaagctggcaccaggg 12065 12064 1 4 SEQ ID NO: 548 tgagcaagtgaagaacttt 4705 4724 SEQ ID NO: 1551 aaagccattcagtctctca 10202 10221 1 4 SEQ ID NO: 549 atttgaaatccaaagaagt 4737 4756 SEQ ID NO: 1552 actttctaaacttgaaatt 8175 8195 1 4 SEQ ID NO: 550 atccaaagaagtcccggaa 4810 4829 SEQ ID NO: 1553 tccggggaaacctgggatt 7922 7941 1 4 SEQ ID NO: 551 agagcctacctccgcatct 4812 4831 SEQ ID NO: 1554 agatggtacgttagcctct 8732 8751 1 4 SEQ ID NO: 552 aatgcctttgaactcccca 4865 4884 SEQ ID NO: 1555 tgggaactacaattcattt 6836 6854 1 4 SEQ ID NO: 553 gaagtccaaattccggatt 4901 4920 SEQ ID NO: 1556 aatcttcaatttattcttc 9342 9361 1 4 SEQ ID NO: 554 tgcaagcagaagccagaag 4905 4924 SEQ ID NO: 1557 cttcaggttccatcgtgca 8603 8622 1 4 SEQ ID NO: 555 gaagagaagattgaatttg 4968 4987 SEQ ID NO: 1558 caaaacctactgtgcttcc 12264 12283 1 4 SEQ ID NO: 556 atgctaaaggcacatatgg 4976 4995 SEQ ID NO: 1559 ccatatgaaagtcaagcat 10801 10820 1 4 SEQ ID NO: 557 tccctcacctccacctctg 4980 4999 SEQ ID NO: 1560 cagattctcagatgaggga 10034 10053 1 4 SEQ ID NO: 558 atttacagctctgacaagt 4961 5000 SEQ ID NO: 1561 acttttctaaacttgaaat 10033 10052 1 4 SEQ ID NO: 559 aggagcctaccaaaataat 4982 5001 SEQ ID NO: 1562 attatgttgaaaacagtct 4725 4744 1 4 SEQ ID NO: 560 aaagctgaagcacatcaat 5066 5105 SEQ ID NO: 1563 attgttgctcatctccttt 12259 12278 1 4 SEQ ID NO: 561 ctgctggaaacaacgagaa 5092 5111 SEQ ID NO: 1564 ttctgattaccaccagcag 12508 12525 1 4 SEQ ID NO: 562 ttgaaggaattcttgaaaa 5106 5125 SEQ ID NO: 1565 ttttaaaagaaatcttcaa 7726 7745 1 4 SEQ ID NO: 563 gaagtaaaagaaaattttg 5143 5162 SEQ ID NO: 1566 caaaacctactgtctcttc 13571 13590 1 4 SEQ ID NO: 564 tgaagaagatggcaaattt 5170 5189 SEQ ID NO: 1567 aaatgtcagctcttgttca 8641 8660 1 4 SEQ ID NO: 565 aggatctgagttatttttc 5199 5216 SEQ ID NO: 1568 gcaagtcagcccagttcct 9719 9738 1 4 SEQ ID NO: 566 gtgcccttctcggttgctg 5257 5276 SEQ ID NO: 1569 cagccattgacatgagcac 5830 5849 1 4 SEQ ID NO: 567 ggcgcggcctgcgctgctg 5281 5300 SEQ ID NO: 1570 cagctccacagactccgcc 6985 7004 1 4 SEQ ID NO: 568 ctgcgctgctgctgctgct 5316 5335 SEQ ID NO: 1571 agcagaaggtgcgaagcag 7247 7266 1 4 SEQ ID NO: 569 gctggtggcgggcgccagg 5317 5336 SEQ ID NO: 1572 cctggattccacatgcagc 7246 7265 1 4 SEQ ID NO: 570 aagaggaaatgctggaaaa 5333 5352 SEQ ID NO: 1573 tttttcttcactacatctt 7615 7635 1 4 SEQ ID NO: 571 ctggaaaatgtcagcctgg 5338 5357 SEQ ID NO: 1574 ccagacttccacatcccag 12934 12953 1 4 SEQ ID NO: 572 tggagtccctgggactgct 5399 5418 SEQ ID NO: 1575 agcatgcctagtttctcca 8372 8391 1 4 SEQ ID NO: 573 ggagtccctgggactgctg 5404 5423 SEQ ID NO: 1576 cagcatgcctagtttctcc 6496 8515 1 4 SEQ ID NO: 574 tgggactgctgattcaaga 5437 5456 SEQ ID NO: 1577 tcttccatcacttgaccca 9685 9704 1 4 SEQ ID NO: 575 ctgctgattcaagaagtgc 5442 5461 SEQ ID NO: 1578 gcacaccttgacattgcag 4421 4440 1 4 SEQ ID NO: 576 tgccaccaggatcaactgc 5458 5477 SEQ ID NO: 1579 gcaggctgaactggtggca 6380 8399 1 4 SEQ ID NO: 577 gccaccaggatcaactgca 5466 5485 SEQ ID NO: 1580 tgcaggctgaactggtggc 6417 6436 1 4 SEQ ID NO: 578 tgcaaggttgagcgtggag 5486 5506 SEQ ID NO: 1581 cctccatcctctgatctga 11812 11831 1 4 SEQ ID NO: 579 caaggttgagctggaggtt 5498 5517 SEQ ID NO: 1582 aacccctacatgaagcttg 7024 7043 1 4 SEQ ID NO: 580 ctctgcagcttcatcctga 5499 5518 SEQ ID NO: 1583 tcaggaagcttctcaagag 10428 10445 1 4 SEQ ID NO: 581 cagcttcatcctgaagaac 5504 5523 SEQ ID NO: 1584 ggtcttgagttaaatgctg 6123 6142 1 4 SEQ ID NO: 582 gcttcatcctgaagaccag 5576 5595 SEQ ID NO: 1585 ctggacgctaagaggaagc 10767 10776 1 4 SEQ ID NO: 583 tcatcctgaagaccagcca 5649 5668 SEQ ID NO: 1586 tggcatggcattatgatga 12756 12775 1 4 SEQ ID NO: 584 gaaaaccaagaactctgag 5684 5703 SEQ ID NO: 1587 ctcaaccttaatgattttc 9005 9025 1 4 SEQ ID NO: 585 agaactctgaggagtttgc 5767 5785 SEQ ID NO: 1588 gcaagctatatcagtattc 9429 6448 1 4 SEQ ID NO: 586 tctgaggagtttgctgcag 5768 5787 SEQ ID NO: 1589 ctgcagggatcccccagat 13304 13323 1 4 SEQ ID NO: 587 tttgctgcagccatgtcca 5640 5859 SEQ ID NO: 1590 tggaagtgtcagtggcaaa 11168 11187 1 4 SEQ ID NO: 588 caagaggggcatcatttct 5885 5907 SEQ ID NO: 1591 agaataaatgacgttcttg 10911 10930 1 4 SEQ ID NO: 589 tcactttaccgtcaagacg 6035 6054 SEQ ID NO: 1592 cgtctacactatcatgtga 8333 8352 1 4 SEQ ID NO: 590 tttaccgtcaagacgagga 6045 6064 SEQ ID NO: 1593 tccttgacatgttgataaa 11213 11232 1 4 SEQ ID NO: 591 cactggacgctaagaggaa 8080 6099 SEQ ID NO: 1594 ttccagaaagcagccagtg 9372 9391 1 4 SEQ ID NO: 592 aggaagcatgtggcagaag 8081 6100 SEQ ID NO: 1595 cttcatacacattaatcct 10660 10679 1 4 SEQ ID NO: 593 caaggagcaacacctcttc 1639 6158 SEQ ID NO: 1596 gaagtagtactgcatgttg 7931 7950 1 4 SEQ ID NO: 594 acagactttgaaacttgaa 6193 6212 SEQ ID NO: 1597 ttcaattcttcaatgctgt 8670 8889 1 4 SEQ ID NO: 595 tgatgaagcagtcacatct 6233 6252 SEQ ID NO: 1598 agatttgaggattccatca 10201 10220 1 4 SEQ ID NO: 596 agcagtcacatctctcttg 6259 6288 SEQ ID NO: 1599 caaggagaaactgactcgt 9056 9075 1 4 SEQ ID NO: 597 ccagccccatcactttaca 6277 6296 SEQ ID NO: 1600 tgtagtctcctggtgctgg 13310 13329 1 4 SEQ ID NO: 598 ctccactccatcctccagg 6279 6298 SEQ ID NO: 1601 ctggagcttagtaatggag 8413 8432 1 4 SEQ ID NO: 599 catgccaacccccttctga 6312 6331 SEQ ID NO: 1602 tcagatgagggaacacatg 10719 10738 1 4 SEQ ID NO: 600 gagagatcttcaacatggc 6313 6332 SEQ ID NO: 1603 gccaccctggaactctctc 8985 8904 1 4 SEQ ID NO: 601 tcaacatggcgagggatca 6338 6357 SEQ ID NO: 1604 tgatcccacctctcattga 8520 8539 1 4 SEQ ID NO: 602 ccaccttgtatgcgctgag 6344 6363 SEQ ID NO: 1605 ctcagggatctgaaggtgg 11700 11719 1 4 SEQ ID NO: 603 gtcaacaactatcataaga 6372 6391 SEQ ID NO: 1606 tcttgagttaaatgctgac 10427 10446 1 4 SEQ ID NO: 604 tggacattgctaattacct 6407 6426 SEQ ID NO: 1607 aggtatattcgaaagtcca 7991 8010 1 4 SEQ ID NO: 605 ggacattgctaattacctg 6412 6431 SEQ ID NO: 1608 caggtatattcgaaagtcc 11479 11498 1 4 SEQ ID NO: 606 ttctgcgggtcattggaaa 6457 6476 SEQ ID NO: 1609 tttcacatgccaaggagaa 10052 10071 1 4 SEQ ID NO: 607 ccagaactcaagtcttcaa 6486 6505 SEQ ID NO: 1610 ttgaagtgtatgtctcctg 11363 11382 1 4 SEQ ID NO: 608 agtcttcaatcctgaaatg 6487 6506 SEQ ID NO: 1611 catttctgattggtggact 11362 11381 1 4 SEQ ID NO: 609 tgagcaagtgaagaacttt 6532 6551 SEQ ID NO: 1612 aaagtgccacttttactca 7979 7998 1 4 SEQ ID NO: 610 agcaagtgaagaactttgt 6550 6569 SEQ ID NO: 1613 acaaagtcagtgccctgct 9042 9061 1 4 SEQ ID NO: 611 tctgaaagaatctcaactt 6603 6622 SEQ ID NO: 1614 aagtccataatggttcaga 6803 6822 1 4 SEQ ID NO: 612 actgtcatggacttcagaa 6886 6705 SEQ ID NO: 1615 ttctgaatatattgtcagt 6708 6727 1 4 SEQ ID NO: 613 acttgacccagcctcagcc 6688 6707 SEQ ID NO: 1616 ggctcaccctgagagaagt 10433 10452 1 4 SEQ ID NO: 614 tccaaataactaccttcct 6702 6721 SEQ ID NO: 1617 aggaagatatgaagatgga 10151 10170 1 4 SEQ ID NO: 615 actaccctcactgcctttg 6729 6748 SEQ ID NO: 1618 caaatttgtggagggtagt 11153 11172 1 4 SEQ ID NO: 616 ttggatttgcttcagctga 6754 6773 SEQ ID NO: 1619 tcagtataagtacaaccaa 7273 7292 1 4 SEQ ID NO: 617 ttggaagctctttttggga 6815 6834 SEQ ID NO: 1620 tcccgattcacgcttccaa 11396 11415 1 4 SEQ ID NO: 618 ggaagctctttttgggaag 6906 6925 SEQ ID NO: 1621 cttcagaaagctaccttcc 10691 10710 1 4 SEQ ID NO: 619 tttttcccagacagtgtca 6965 6984 SEQ ID NO: 1622 tgaccttctctaagcaaaa 10563 10682 1 4 SEQ ID NO: 620 agacagtgtcaacaaagct 7051 7070 SEQ ID NO: 1623 agcttggttttgccagtct 7096 7115 1 4 SEQ ID NO: 621 ctttggctataccaaagat 7092 7111 SEQ ID NO: 1624 atctcgtgtctaggaaaag 8413 8432 1 4 SEQ ID NO: 622 caaagatgataaacatgag 7191 7210 SEQ ID NO: 1625 ctcaaggataacgtgtttg 7766 7787 1 4 SEQ ID NO: 623 gatatggtaaatggaataa 7219 7238 SEQ ID NO: 1626 ttatcttataattatatca 8518 8537 1 4 SEQ ID NO: 624 ggaataatgctcagtgttg 7224 7243 SEQ ID NO: 1627 caaacacttacttgaattc 10293 10312 1 4 SEQ ID NO: 625 tttgaaatccaaagaagtc 7285 7304 SEQ ID NO: 1628 gacttcagagaaatacaaa 7312 7331 1 4 SEQ ID NO: 626 gatcccccagatgattgga 7319 7335 SEQ ID NO: 1629 tccaatttccctgtggatc 13201 13220 1 4 SEQ ID NO: 627 cagatattggagaggtcaa 7357 7376 SEQ ID NO: 1630 tgaccacacaaaacagtct 7246 7265 1 4 SEQ ID NO: 628 agaatgacttttttctctc 7386 7405 SEQ ID NO: 1631 tgaagtccggattcattct 9715 9734 1 4 SEQ ID NO: 629 gaactccccactggagctg 7456 7475 SEQ ID NO: 1632 cagctcaaccgtacagttc 9068 9087 1 4 SEQ ID NO: 630 atatcttcatgggagtcaa 7467 7485 SEQ ID NO: 1633 tgacttcagtgcagaatat 8517 8536 1 4 SEQ ID NO: 631 gtcatgctccccggaggca 7484 7503 SEQ ID NO: 1634 tggccccgtttaccatgac 10637 1656 1 4 SEQ ID NO: 632 gctgaagtttatcattcct 7570 7569 SEQ ID NO: 1635 aggaggctttaagttcagc 10942 10961 1 4 SEQ ID NO: 633 attccttccccaaagagac 7573 7592 SEQ ID NO: 1636 gtctcttcctccatggaat 10741 10760 1 4 SEQ ID NO: 634 ctcattgagaacaggcagt 7607 7626 SEQ ID NO: 1637 actgactgcacgctttgag 9472 9491 1 4 SEQ ID NO: 635 ttgagcagtattctgtcag 7731 7750 SEQ ID NO: 1638 cttagagaagtgtcttcaa 7857 7876 1 4 SEQ ID NO: 636 accttgtccagtgaagtcc 7862 7881 SEQ ID NO: 1639 ggacggtactgtcccaggt 8914 8933 1 4 SEQ ID NO: 637 ccagtgaagtccaaattcc 7865 7884 SEQ ID NO: 1640 ggaaggcagagtttactgg 6000 6019 1 4 SEQ ID NO: 638 acattcagaacaagaaaat 7666 7885 SEQ ID NO: 1641 atttcctaaagctggatgc 5999 6018 1 4 SEQ ID NO: 639 gaaaaatcaagggtgttat 7901 7920 SEQ ID NO: 1642 ataaactgcaagatttttc 12572 12591 1 4 SEQ ID NO: 640 aaatcaagggtgttatttc 7972 7991 SEQ ID NO: 1643 gaaacaatgcattagattt 13214 13233 1 4 SEQ ID NO: 641 tggcattatgatgaagaga 8042 8061 SEQ ID NO: 1644 tctcccgtgtataatgcca 9410 9429 1 4 SEQ ID NO: 642 aagagaagattgaatttga 8126 8147 SEQ ID NO: 1645 tcaaaacctactgtctcct 12430 12449 1 4 SEQ ID NO: 643 aaatgacttccaatttccc 8218 8237 SEQ ID NO: 1646 gggaactacaatttcattt 12293 12312 1 4 SEQ ID NO: 644 atgacttccaatttccctg 8291 8310 SEQ ID NO: 1647 caggctgattacgagtcat 12571 12590 1 4 SEQ ID NO: 645 acttccaatttccctgtgg 8320 8339 SEQ ID NO: 1648 ccacgaaaaatatggaagt 10018 10037 1 4 SEQ ID NO: 646 agttgcaatgagctcatgg 8430 8449 SEQ ID NO: 1649 ccatcagttcagataaact 9997 10016 1 4 SEQ ID NO: 647 tttgcaagaccacctcaat 8431 8450 SEQ ID NO: 1650 attgacctgtccattcaaa 9237 9256 1 4 SEQ ID NO: 648 gaaggagttcaacctccag 8450 9469 SEQ ID NO: 1651 ctggaattgtcattccttc 9283 9302 1 4 SEQ ID NO: 649 acttccacatcccagaaaa 8468 8487 SEQ ID NO: 1652 ttttaacaaaaagtggaag 10964 10983 1 4 SEQ ID NO: 650 ctcttcttaaaaagcgatg 8543 8562 SEQ ID NO: 1653 catcactgccaaaggagag 13493 13512 1 4 SEQ ID NO: 651 aaaagcgatggccgggtcc 8584 8603 SEQ ID NO: 1654 tgactcactcattgatttt 13780 13799 1 4 SEQ ID NO: 652 ttcctttgccttttggtgg 8644 8863 SEQ ID NO: 1655 ccacaaaacaatgaaggga 13186 13205 1 4 SEQ ID NO: 653 caagtctgtgggattccat 8721 8740 SEQ ID NO: 1656 atgggaaaaaacaggcttg 9223 9242 1 4 SEQ ID NO: 654 aagtccctacttttaccat 8733 8752 SEQ ID NO: 1657 atgggaagtataagaactt 4811 4830 1 4 SEQ ID NO: 655 tgcctctcctgggtgttct 8779 8795 SEQ ID NO: 1658 agaaaaacaaacaaacgga 11164 11183 1 4 SEQ ID NO: 656 accagcacagaccatttca 8787 8808 SEQ ID NO: 1659 tgaagtgtagtctcctggt 12595 12614 1 4 SEQ ID NO: 657 ccagcacagaccatttcag 8791 8810 SEQ ID NO: 1660 ctgaaatacaatgctctgg 9082 9101 1 4 SEQ ID NO: 658 actatcatgtgatgggtct 8806 8825 SEQ ID NO: 1661 agacacctgattttatagt 11002 11021 1 4 SEQ ID NO: 659 accacagatgtctgcttca 8936 8955 SEQ ID NO: 1662 tgaaggctgactctgtggt 12809 12828 1 4 SEQ ID NO: 660 ccacagagtgtctgcttcg 8960 8979 SEQ ID NO: 1663 ctgagcaacaatttgtgga 12284 12303 1 4 SEQ ID NO: 661 tttggactccaaaaagaaa 8980 8999 SEQ ID NO: 1664 tttctctcatgattacaaa 11628 11645 1 4 SEQ ID NO: 662 tcaaagaagtcaagattga 8984 9003 SEQ ID NO: 1665 tcaaggataactgtttgag 13853 13902 1 4 SEQ ID NO: 663 atgagaactacgagctgac 8995 9004 SEQ ID NO: 1666 gtcagatattgttgctcat 13882 13901 1 4 SEQ ID NO: 664 ttaaactctgacaccaatg 9033 9052 SEQ ID NO: 1667 cattcattgaagatgttaa 12247 12266 1 4 SEQ ID NO: 665 gaagtataagaactttgcc 9037 9056 SEQ ID NO: 1668 ggcaaatttgaaggacttc 11659 11676 1 4 SEQ ID NO: 666 aagtataagaaactttgca 9051 9070 SEQ ID NO: 1669 tggcaaatttgaaggactt 11645 11684 1 4 SEQ ID NO: 667 ttcttcagcctgctttctg 9121 9140 SEQ ID NO: 1670 cagaatccagatacaagaa 9741 9750 1 4 SEQ ID NO: 668 ctggatcactaaattccca 9124 9143 SEQ ID NO: 1671 tgggtctttccagagcccg 11069 11088 1 4 SEQ ID NO: 669 aaattaatagtggtgctca 9254 9273 SEQ ID NO: 1672 tgagaagccccaagaattt 7969 7988 1 4 SEQ ID NO: 670 ggccattccagaagggaag 9271 9290 SEQ ID NO: 1673 cttccgttctgtaatggcc 9647 7668 1 4 SEQ ID NO: 671 tgccatctcgagagttcca 9324 9343 SEQ ID NO: 1674 tggaactctctccatggca 12826 12847 1 4 SEQ ID NO: 672 catgtcaaacactttgtta 9424 9443 SEQ ID NO: 1675 taacaaattccttgacatg 13600 13619 1 4 SEQ ID NO: 673 tttgttataaatcttattg 9391 9610 SEQ ID NO: 1676 caataagatcaatagcaaa 11057 11075 1 4 SEQ ID NO: 674 tctggaaaaagggtcatga 9640 9659 SEQ ID NO: 1677 tccatgtcccatttacaga 9682 9701 1 4 SEQ ID NO: 675 cagctcttgttcaggtcca 9646 9665 SEQ ID NO: 1678 tggacctgcaccaaagctg 9272 9291 1 4 SEQ ID NO: 676 ggaggttccccagctctgc 9659 9676 SEQ ID NO: 1679 gcagccctgggaaaactcc 10963 10932 1 4 SEQ ID NO: 677 ctgttttgaagactctcca 9732 9751 SEQ ID NO: 1680 tggagggtagtcataacag 12925 12944 1 4 SEQ ID NO: 678 agtggctgaaacgtgtgca 9749 9768 SEQ ID NO: 1681 tgcagagctttctgccact 10271 10290 1 4 SEQ ID NO: 679 ccaaaatagaagggaatct 9609 9826 SEQ ID NO: 1682 agattcctttctttttcaa 11430 11449 1 4 SEQ ID NO: 680 tgaagagaagattgaattt 9855 9874 SEQ ID NO: 1683 aaattctcttttattttca 8802 8821 1 4 SEQ ID NO: 681 agtggtggcaacaccagca 9882 9901 SEQ ID NO: 1684 tgctagtgaggccaacact 9038 9057 1 4 SEQ ID NO: 682 aaggctccacaagtcatca 9956 9975 SEQ ID NO: 1685 tgatgatatctggaacctt 6885 6904 1 4 SEQ ID NO: 683 gtccgccaggtttctagca 9957 9976 SEQ ID NO: 1686 tgctaagaaccttactgac 6884 5903 1 4 SEQ ID NO: 684 tgatatctggaaccttgga 10011 10030 SEQ ID NO: 1687 ttcactgttcctgaaatca 13297 13316 1 4 SEQ ID NO: 685 gtcaagttgagcaatttct 10169 10188 SEQ ID NO: 1688 agaaaaggcacaccttgac 11176 11197 1 4 SEQ ID NO: 686 atccagatggaaaagggaa 10205 10225 SEQ ID NO: 1689 ttccaatttccctgtggat 13764 13783 1 4 SEQ ID NO: 687 atttgtttgtcaaagaagt 10226 10245 SEQ ID NO: 1690 acttcagagagaaatacat 12072 12091 1 4 SEQ ID NO: 688 ctggaaaatgtcagcctgg 10232 10251 SEQ ID NO: 1691 ccagacttcagttaccagc 11658 11677 1 4 SEQ ID NO: 689 accaggaggttcttcttca 1301 10320 SEQ ID NO: 1692 tgaagtgtgatctcctggt 10539 10558 1 4 SEQ ID NO: 690 aaagaagttctgaaaagat 10400 10419 SEQ ID NO: 1693 attccatcacaaaatcctt 13963 13982 1 4 SEQ ID NO: 691 gctacagcttatggctcca 10416 10435 SEQ ID NO: 1694 tggatctaaatgcagtagc 13208 13227 1 4 SEQ ID NO: 692 atcaatatgatcaatttgt 10451 10480 SEQ ID NO: 1695 caaagaaatcaagattgat 12583 12602 1 4 SEQ ID NO: 693 gaattatctttaaaacatt 10575 10594 SEQ ID NO: 1696 atgtggacaaatataccgg 12355 12374 1 4 SEQ ID NO: 694 cgaggcccgcgctgctggc 10910 10929 SEQ ID NO: 1697 gccagaagtgagatcctcg 4993 5012 1 4 SEQ ID NO: 695 acaactatgaggctgagag 10918 10937 SEQ ID NO: 1698 ctctgagcaacaaattttt 13994 14013 1 4 SEQ ID NO: 696 gctgagagttccagtggag 10975 10994 SEQ ID NO: 1699 ctccatggcaaatgtcagg 8518 8537 1 4 SEQ ID NO: 697 tgaagaaaaaccaagaact 10996 11015 SEQ ID NO: 1700 gagtcattgaggttcttca 12165 12184 1 4 SEQ ID NO: 698 cctacttacatcctgaaca 11256 11275 SEQ ID NO: 1701 tgttcataagggaggtagg 11624 11643 1 4 SEQ ID NO: 699 ctacttacatcctgaacat 11280 11299 SEQ ID NO: 1702 atgttcataagggaggtag 9481 9500 1 4 SEQ ID NO: 700 gagacagaagccaagagcc 11282 11301 SEQ ID NO: 1703 gcttggttttgcccagtct 9039 9058 1 4 SEQ ID NO: 701 cactcactttaccgtcaag 11308 11327 SEQ ID NO: 1704 cttgaacaccaaagtcact 11027 11046 1 4 SEQ ID NO: 702 ctgatcagcagcagccagt 11406 11424 SEQ ID NO: 1705 actgggaagtgcttatcag 8865 8884 1 4 SEQ ID NO: 703 actggacgctaagaggaag 11472 11491 SEQ ID NO: 1706 cttccccaaagagaggagt 12298 12317 1 4 SEQ ID NO: 704 agaggaagcatgtggcaga 11537 11556 SEQ ID NO: 1707 tctggcatttactttctct 13715 13734 1 4 SEQ ID NO: 705 tgaagactctccaggaact 11727 11746 SEQ ID NO: 1708 agttgaaggagactattca 12094 12113 1 4 SEQ ID NO: 706 ctctgagcaaaatatccag 11787 11806 SEQ ID NO: 1709 ctggttactgagctgagag 13096 13115 1 4 SEQ ID NO: 707 atgaagcagtcacatctct 11851 11870 SEQ ID NO: 1710 agagctgccagtcttcatt 6246 6265 1 4 SEQ ID NO: 708 ttgccacagctgattgagg 11964 12003 SEQ ID NO: 1711 cctcctacagtggtggcaa 9212 9231 1 4 SEQ ID NO: 709 agctgattgaggtgtccag 12042 12061 SEQ ID NO: 1712 ctggattccacatgcagct 12661 12680 1 4 SEQ ID NO: 710 tgctccactcacatcctcc 12135 12154 SEQ ID NO: 1713 ggaggctttaagttcagca 8063 8062 1 4 SEQ ID NO: 711 tgaaacgtgtgcatgtcaa 12171 12190 SEQ ID NO: 1714 ttgggagagacaagtttca 10080 10099 1 4 SEQ ID NO: 712 gacattgctaattacctga 12211 12230 SEQ ID NO: 1715 tcagaagctaagcaatgtc 6985 7004 1 4 SEQ ID NO: 713 ttcttcttcagactttcct 12420 12439 SEQ ID NO: 1716 aggagagtccaaatttaga 13439 13458 1 4 SEQ ID NO: 714 ccaatatcttgaaactcag 12439 1258 SEQ ID NO: 1717 tctgaattcattcaattgg 14076 14097 1 4 SEQ ID NO: 715 aaagttagtgaaaagaagt 12440 12459 SEQ ID NO: 1718 aactaccctcactgccttt 14077 14096 1 4 SEQ ID NO: 716 aagttagtgaaagaagttc 12468 12486 SEQ ID NO: 1719 gaacctctggcattttact 13384 13403 1 4 SEQ ID NO: 717 aaagaagttctgaaagaat 12889 12908 SEQ ID NO: 1720 attctctggtaactacttt 13633 13652 1 4 SEQ ID NO: 718 tttggctataccaaagatg 13031 13050 SEQ ID NO: 1721 catcttaggcactgacaaa 14044 14063 1 4 SEQ ID NO: 719 tgttgagaagctgattaaa 13087 13106 SEQ ID NO: 1722 tttagccatcggctcaaca 1392 13811 1 4 SEQ ID NO: 720 caggaagggctcaaagaat 13143 13162 SEQ ID NO: 1723 attcctttaacaattcctg 7024 7043 1 4 SEQ ID NO: 721 aggaagggctcaaagaatg 13182 13201 SEQ ID NO: 1724 cattcctttaacaattcct 10160 10179 1 4 SEQ ID NO: 722 gaagggctcaaagaatgac 13318 13337 SEQ ID NO: 1725 gtcagtcttcaggctcttc 12399 12418 1 4 SEQ ID NO: 723 caaagaatgacttttttct 13369 13388 SEQ ID NO: 1726 agaaggatggcattttttg 10729 10746 1 4 SEQ ID NO: 724 catggagaatgcctttgaa 13443 13462 SEQ ID NO: 1727 ttcagagccaaagtccatg 11480 11499 1 4 SEQ ID NO: 725 ggagccaaggctggagtaa 13552 13571 SEQ ID NO: 1728 ttactccaacgccagctcc 10095 10114 1 4 SEQ ID NO: 726 tcattccttccccaaagag 13599 13618 SEQ ID NO: 1729 ctctctggggcatctatgc 13855 13884 1 4 SEQ ID NO: 727 acctatgagctccagagag 13629 13648 SEQ ID NO: 1730 ctctcaagaccacagaagg 6428 6447 1 4 SEQ ID NO: 728 gggcaaaaacgtcttacag 13718 13737 SEQ ID NO: 1731 tctgaaagacaacgtgccc 9944 9963 1 4 SEQ ID NO: 729 accctggaccttcagaaca 13765 13784 SEQ ID NO: 1732 tgttgctaaggttcagggt 10205 10224 1 4 SEQ ID NO: 730 atgggcgacctaagttgtg 13947 13966 SEQ ID NO: 1733 cacaaattagtttcaccat 7848 7867 1 4 SEQ ID NO: 731 gatgaagagaagattgaat 14050 14069 SEQ ID NO: 1734 atccagcttccccacactt 4222 4241 1 4 SEQ ID NO: 732 caatgtgataccaaaaaaa 4404 4423 SEQ ID NO: 1735 tttttggaaatgccattgt 9595 9614 3 3 SEQ ID NO: 733 gtagataccaaaaaaatga 4543 4562 SEQ ID NO: 1736 tcatgtgatgggtctctac 9055 9074 3 3 SEQ ID NO: 734 gcttcagttcatttggact 25 44 SEQ ID NO: 1737 agtcaagaaggacttaagc 9228 9247 2 3 SEQ ID NO: 735 tttgtttgtcaaagaagtc 39 58 SEQ ID NO: 1738 gacttcagagaaatacaaa 11177 11196 2 3 SEQ ID NO: 736 ttgtttgtcaaagaagtca 219 23 SEQ ID NO: 1739 tgacttcagagaaatacaa 8126 8145 2 3 SEQ ID NO: 737 tggcaatgggaaactcgct 283 302 SEQ ID NO: 1740 agcgagagtcccctgccat 7457 7479 2 3 SEQ ID NO: 738 aacctctggcatttacttt 396 415 SEQ ID NO: 1741 aaaggagatgtcaagggtt 8969 8988 2 3 SEQ ID NO: 739 catttactttctctcatga 464 483 SEQ ID NO: 1742 tcatttgaaagaataaatg 11391 11410 2 3 SEQ ID NO: 740 aaagtcagtgccctgctta 574 593 SEQ ID NO: 1743 taagaaccttactgacttt 7042 7061 2 3 SEQ ID NO: 741 tcccattttttgagacctt 822 841 SEQ ID NO: 1744 aaggacttcaggaatggga 8801 8820 2 3 SEQ ID NO: 742 catcaatattgatcaattt 857 876 SEQ ID NO: 1745 aaattaaaaagtcttgatg 11346 11365 2 3 SEQ ID NO: 743 taaagatagttatgattta 1079 1098 SEQ ID NO: 1746 taaaccaaaaacttggtta 12403 12422 2 3 SEQ ID NO: 744 tattgatgaaatcattgaa 1105 1124 SEQ ID NO: 1747 ttcaaagacttaaaaaata 13104 13123 2 3 SEQ ID NO: 745 atgatctacatttgtttat 1168 1167 SEQ ID NO: 1748 ataaagaaattaaagtcat 3027 3046 2 3 SEQ ID NO: 746 agagacacatacagaatat 1303 1322 SEQ ID NO: 1749 atatattgtcagtgcctct 7848 7867 2 3 SEQ ID NO: 747 gacacatacagaatataga 1432 1451 SEQ ID NO: 1750 tctaaattcagttcttgtc 9130 9149 2 3 SEQ ID NO: 748 agcatgtcaaacactttgt 1492 1511 SEQ ID NO: 1751 acaaagtcagtgccctgct 10920 10939 2 3 SEQ ID NO: 749 tttttagaggaaaccaagg 1567 1586 SEQ ID NO: 1752 ccttgtgtacaccaaaaac 7420 7439 2 3 SEQ ID NO: 750 ttttagaggaaaccaaggc 1619 1638 SEQ ID NO: 1753 gcctttgtgtacaccaaaa 5512 5531 2 3 SEQ ID NO: 751 ggaagatagacttcctgaa 1736 1755 SEQ ID NO: 1754 ttcagaaatactgtttttc 10447 10466 2 3 SEQ ID NO: 752 cactgtttctgagtcccag 1802 1821 SEQ ID NO: 1755 ctgggacctaccaagagtg 13726 13745 2 3 SEQ ID NO: 753 cacaaatcctttggctgtg 1933 1962 SEQ ID NO: 1756 cacatttcaaggaattgtg 3544 3563 2 3 SEQ ID NO: 754 ttcctggatacactgttcc 1948 1967 SEQ ID NO: 1757 ggaactgttgactcaggac 8267 8286 2 3 SEQ ID NO: 755 gaaatctcaagctttctct 2076 2095 SEQ ID NO: 1758 agagccaggtcgagctttc 10459 10476 2 3 SEQ ID NO: 756 tttcttcatcttcatctgt 2213 2232 SEQ ID NO: 1759 acagctgaaagagatgaaa 8316 8335 2 3 SEQ ID NO: 757 tctaccgctaaaggagcag 2366 2385 SEQ ID NO: 1760 ctgcacgctttgaggtaga 9648 9667 2 3 SEQ ID NO: 758 ctaccgctaaaggagcagt 2400 2419 SEQ ID NO: 1761 actgcacgctttgaggtcg 10029 10048 2 3 SEQ ID NO: 759 aggggcctcttttcaccaa 2409 2428 SEQ ID NO: 1762 ttggccaggaagtggccct 4943 4962 2 3 SEQ ID NO: 760 tctccatccctgtaaaaag 2562 2561 SEQ ID NO: 1763 ctttttcaccaacggagaa 10012 10031 2 3 SEQ ID NO: 761 gaaaaacaaagcagattat 2575 2594 SEQ ID NO: 1764 ataaactgcaagatttttc 12152 12171 2 3 SEQ ID NO: 762 actcactcattgattttct 2757 2776 SEQ ID NO: 1765 agaaaatcaggatcgtagt 11807 11626 2 3 SEQ ID NO: 763 taaactaatagatgtaatc 3244 3263 SEQ ID NO: 1766 gattaccaccagcagttta 10895 10914 2 3 SEQ ID NO: 764 caaaaacgagcttcggaag 3660 6379 SEQ ID NO: 1767 cttcgtgagaatatttgaa 11442 11481 2 3 SEQ ID NO: 765 tggaataatgctcagtgtt 6373 3692 SEQ ID NO: 1768 aacacttaacttgaattca 12919 12938 2 3 SEQ ID NO: 766 gatttgaaatccaaagaag 3675 3694 SEQ ID NO: 1769 cttcagagaaatacaaatc 13777 13796 2 3 SEQ ID NO: 767 atttgaaatccaaagaagt 4095 4115 SEQ ID NO: 1770 acttcagagaaatacaaat 9539 9558 2 3 SEQ ID NO: 768 atcaacagccgcttctttg 4543 9583 SEQ ID NO: 1771 caaagaagtcaagattgat 8239 8258 2 3 SEQ ID NO: 769 tgttttgaagactctccag 5127 5146 SEQ ID NO: 1772 ctggaaagttaaaacaaac 13510 13529 2 3 SEQ ID NO: 770 cccttctgatagatgtggt 5345 5364 SEQ ID NO: 1773 accaaagctggcaccaggg 13688 13885 2 3 SEQ ID NO: 771 tgagcaagtgaagaacttt 5412 5431 SEQ ID NO: 1774 aaagccattcagtctctca 8772 8791 2 3 SEQ ID NO: 772 atttgaaatccaaagaagt 5504 5523 SEQ ID NO: 1775 actttctaaacttgaaatt 8786 8805 2 3 SEQ ID NO: 773 atccaaagaagtcccggaa 5840 5859 SEQ ID NO: 1776 tccggggaaacctgggatt 11221 11240 2 3 SEQ ID NO: 774 agagcctacctccgcatct 6277 6295 SEQ ID NO: 1777 agatggtacgttagcctct 13648 13667 2 3 SEQ ID NO: 775 aatgcctttgaactcccca 6280 6299 SEQ ID NO: 1778 tgggaactacaattcattt 8531 8550 2 3 SEQ ID NO: 776 gaagtccaaattccggatt 6312 6331 SEQ ID NO: 1779 aatcttcaatttattcttc 10748 10767 2 3 SEQ ID NO: 777 tgcaagcagaagccagaag 6480 6499 SEQ ID NO: 1780 cttcaggttccatcgtgca 7154 7173 2 3 SEQ ID NO: 778 gaagagaagattgaatttg 6498 6517 SEQ ID NO: 1781 caaaacctactgtgcttcc 11316 11335 2 3 SEQ ID NO: 779 atgctaaaggcacatatgg 6688 6707 SEQ ID NO: 1782 ccatatgaaagtcaagcat 13433 13452 2 3 SEQ ID NO: 780 tccctcacctccacctctg 7052 7071 SEQ ID NO: 1783 cagattctcagatgaggga 12963 12982 2 3 SEQ ID NO: 781 atttacagctctgacaagt 7348 7367 SEQ ID NO: 1784 acttttctaaacttgaaat 12652 12671 2 3 SEQ ID NO: 782 aggagcctaccaaaataat 7745 7764 SEQ ID NO: 1785 attatgttgaaaacagtct 13268 13287 2 3 SEQ ID NO: 783 aaagctgaagcacatcaat 8042 8061 SEQ ID NO: 1786 attgttgctcatctccttt 9473 9492 2 3 SEQ ID NO: 784 ctgctggaaacaacgagaa 9397 9416 SEQ ID NO: 1787 ttctgattaccaccagcag 14075 14094 2 3 SEQ ID NO: 785 ttgaaggaattcttgaaaa 0 19 SEQ ID NO: 1788 ttttaaaagaaatcttcaa 11583 11582 1 3 SEQ ID NO: 786 gaagtaaaagaaaattttg 17 36 SEQ ID NO: 1789 caaaacctactgtctcttc 12670 12689 1 3 SEQ ID NO: 787 tgaagaagatggcaaattt 39 58 SEQ ID NO: 1790 aaatgtcagctcttgttca 1217 1236 1 3 SEQ ID NO: 788 aggatctgagttatttttc 42 61 SEQ ID NO: 1791 gcaagtcagcccagttcct 3744 3763 1 3 SEQ ID NO: 789 gtgcccttctcggttgctg 64 83 SEQ ID NO: 1792 cagccattgacatgagcac 1355 1374 1 3 SEQ ID NO: 790 ggcgcggcctgcgctgctg 81 100 SEQ ID NO: 1793 cagctccacagactccgcc 2674 2693 1 3 SEQ ID NO: 791 ctgcgctgctgctgctgct 95 115 SEQ ID NO: 1794 agcagaaggtgcgaagcag 1080 1099 1 3 SEQ ID NO: 792 gctggtggcgggcgccagg 192 211 SEQ ID NO: 1795 cctggattccacatgcagc 8301 8320 1 3 SEQ ID NO: 793 aagaggaaatgctggaaaa 229 248 SEQ ID NO: 1796 tttttcttcactacatctt 7077 7096 1 3 SEQ ID NO: 794 ctggaaaatgtcagcctgg 245 264 SEQ ID NO: 1797 ccagacttccacatcccag 10059 10078 1 3 SEQ ID NO: 795 tggagtccctgggactgct 289 308 SEQ ID NO: 1798 agcatgcctagtttctcca 8602 8621 1 3 SEQ ID NO: 796 ggagtccctgggactgctg 308 327 SEQ ID NO: 1799 cagcatgcctagtttctcc 13316 13335 1 3 SEQ ID NO: 797 tgggactgctgattcaaga 325 344 SEQ ID NO: 1800 tcttccatcacttgaccca 10696 10715 1 3 SEQ ID NO: 798 ctgctgattcaagaagtgc 335 354 SEQ ID NO: 1801 gcacaccttgacattgcag 4740 4759 1 3 SEQ ID NO: 799 tgccaccaggatcaactgc 340 359 SEQ ID NO: 1802 gcaggctgaactggtggca 1281 1300 1 3 SEQ ID NO: 800 gccaccaggatcaactgca 365 384 SEQ ID NO: 1803 tgcaggctgaactggtggc 1335 1354 1 3 SEQ ID NO: 801 tgcaaggttgagcgtggag 375 394 SEQ ID NO: 1804 cctccatcctctgatctga 5104 5123 1 3 SEQ ID NO: 802 caaggttgagctggaggtt 377 396 SEQ ID NO: 1805 aacccctacatgaagcttg 2688 2707 1 3 SEQ ID NO: 803 ctctgcagcttcatcctga 391 410 SEQ ID NO: 1806 tcaggaagcttctcaagag 1531 1550 1 3 SEQ ID NO: 804 cagcttcatcctgaagaac 396 415 SEQ ID NO: 1807 ggtcttgagttaaatgctg 5222 5241 1 3 SEQ ID NO: 805 gcttcatcctgaagaccag 419 438 SEQ ID NO: 1808 ctggacgctaagaggaagc 3525 3544 1 3 SEQ ID NO: 806 tcatcctgaagaccagcca 422 441 SEQ ID NO: 1809 tggcatggcattatgatga 2199 2218 1 3 SEQ ID NO: 807 gaaaaccaagaactctgag 423 442 SEQ ID NO: 1810 ctcaaccttaatgattttc 9719 9738 1 3 SEQ ID NO: 808 agaactctgaggagtttgc 443 462 SEQ ID NO: 1811 gcaagctatatcagtattc 9066 9085 1 3 SEQ ID NO: 809 tctgaggagtttgctgcag 445 464 SEQ ID NO: 1812 ctgcagggatcccccagat 5639 5658 1 3 SEQ ID NO: 810 tttgctgcagccatgtcca 475 494 SEQ ID NO: 1813 tggaagtgtcagtggcaaa 2996 3015 1 3 SEQ ID NO: 811 caagaggggcatcatttct 476 495 SEQ ID NO: 1814 agaataaatgacgttcttg 2995 3014 1 3 SEQ ID NO: 812 tcactttaccgtcaagacg 482 501 SEQ ID NO: 1815 cgtctacactatcatgtga 1285 1304 1 3 SEQ ID NO: 813 tttaccgtcaagacgagga 499 518 SEQ ID NO: 1816 tccttgacatgttgataaa 7481 7500 1 3 SEQ ID NO: 814 cactggacgctaagaggaa 518 537 SEQ ID NO: 1817 ttccagaaagcagccagtg 13813 13832 1 3 SEQ ID NO: 815 aggaagcatgtggcagaag 520 539 SEQ ID NO: 1818 cttcatacacattaatcct 7564 7583 1 3 SEQ ID NO: 816 caaggagcaacacctcttc 547 566 SEQ ID NO: 1819 gaagtagtactgcatgttg 4844 4853 1 3 SEQ ID NO: 817 acagactttgaaacttgaa 567 586 SEQ ID NO: 1820 ttcaattcttcaatgctgt 7969 7958 1 3 SEQ ID NO: 818 tgatgaagcagtcacatct 568 587 SEQ ID NO: 1821 agatttgaggattccatca 7965 7987 1 3 SEQ ID NO: 819 agcagtcacatctctcttg 570 589 SEQ ID NO: 1822 caaggagaaactgactcgt 7900 7919 1 3 SEQ ID NO: 820 ccagccccatcactttaca 573 592 SEQ ID NO: 1823 tgtagtctcctggtgctgg 6290 6309 1 3 SEQ ID NO: 821 ctccactccatcctccagg 574 593 SEQ ID NO: 1824 ctggagcttagtaatggag 6289 6308 1 3 SEQ ID NO: 822 catgccaacccccttctga 589 608 SEQ ID NO: 1825 tcagatgagggaacacatg 7408 7124 1 3 SEQ ID NO: 823 gagagatcttcaacatggc 607 626 SEQ ID NO: 1826 gccaccctggaactctctc 10058 10077 1 3 SEQ ID NO: 824 tcaacatggcgagggatca 621 640 SEQ ID NO: 1827 tgatcccacctctcattga 8071 8090 1 3 SEQ ID NO: 825 ccaccttgtatgcgctgag 639 658 SEQ ID NO: 1828 ctcagggatctgaaggtgg 8767 8786 1 3 SEQ ID NO: 826 gtcaacaactatcataaga 655 674 SEQ ID NO: 1829 tcttgagttaaatgctgac 2920 2939 1 3 SEQ ID NO: 827 tggacattgctaattacct 662 681 SEQ ID NO: 1830 aggtatattcgaaagtcca 4532 4551 1 3 SEQ ID NO: 828 ggacattgctaattacctg 672 691 SEQ ID NO: 1831 caggtatattcgaaagtcc 10572 10591 1 3 SEQ ID NO: 829 ttctgcgggtcattggaaa 677 696 SEQ ID NO: 1832 tttcacatgccaaggagaa 2483 2502 1 3 SEQ ID NO: 830 ccagaactcaagtcttcaa 679 698 SEQ ID NO: 1833 ttgaagtgtatgtctcctg 11169 11185 1 3 SEQ ID NO: 831 agtcttcaatcctgaaatg 690 709 SEQ ID NO: 1834 catttctgattggtggact 5956 5975 1 3 SEQ ID NO: 832 tgagcaagtgaagaacttt 691 710 SEQ ID NO: 1835 aaagtgccacttttactca 3507 3528 1 3 SEQ ID NO: 833 agcaagtgaagaactttgt 692 711 SEQ ID NO: 1836 acaaagtcagtgccctgct 3468 2487 1 3 SEQ ID NO: 834 tctgaaagaatctcaactt 694 713 SEQ ID NO: 1837 aagtccataatggttcaga 13366 13375 1 3 SEQ ID NO: 835 actgtcatggacttcagaa 695 714 SEQ ID NO: 1838 ttctgaatatattgtcagt 1801 1820 1 3 SEQ ID NO: 836 acttgacccagcctcagcc 769 788 SEQ ID NO: 1839 ggctcaccctgagagaagt 3011 3030 1 3 SEQ ID NO: 837 tccaaataactaccttcct 770 789 SEQ ID NO: 1840 aggaagatatgaagatgga 3010 3029 1 3 SEQ ID NO: 838 actaccctcactgcctttg 775 794 SEQ ID NO: 1841 caaatttgtggagggtagt 12660 12679 1 3 SEQ ID NO: 839 ttggatttgcttcagctga 815 834 SEQ ID NO: 1842 tcagtataagtacaaccaa 4811 4830 1 3 SEQ ID NO: 840 ttggaagctctttttggga 857 876 SEQ ID NO: 1843 tcccgattcacgcttccaa 9331 9360 1 3 SEQ ID NO: 841 ggaagctctttttgggaag 894 913 SEQ ID NO: 1844 cttcagaaagctaccttcc 13457 13476 1 3 SEQ ID NO: 842 tttttcccagacagtgtca 805 914 SEQ ID NO: 1845 tgaccttctctaagcaaaa 13456 13475 1 3 SEQ ID NO: 843 agacagtgtcaacaaagct 900 919 SEQ ID NO: 1846 agcttggttttgccagtct 4284 4303 1 3 SEQ ID NO: 844 ctttggctataccaaagat 901 920 SEQ ID NO: 1847 atctcgtgtctaggaaaag 1067 1085 1 3 SEQ ID NO: 845 caaagatgataaacatgag 925 944 SEQ ID NO: 1848 ctcaaggataacgtgtttg 6786 6805 1 3 SEQ ID NO: 846 gatatggtaaatggaataa 926 945 SEQ ID NO: 1849 ttatcttataattatatca 1236 1257 1 3 SEQ ID NO: 847 ggaataatgctcagtgttg 946 965 SEQ ID NO: 1850 caaacacttacttgaattc 4584 4603 1 3 SEQ ID NO: 848 tttgaaatccaaagaagtc 947 966 SEQ ID NO: 1851 gacttcagagaaatacaaa 4583 4602 1 3 SEQ ID NO: 849 gatcccccagatgattgga 948 967 SEQ ID NO: 1852 tccaatttccctgtggatc 5091 5110 1 3 SEQ ID NO: 850 cagatattggagaggtcaa 970 989 SEQ ID NO: 1853 tgaccacacaaaacagtct 7979 7998 1 3 SEQ ID NO: 851 agaatgacttttttctctc 1000 1019 SEQ ID NO: 1854 tgaagtccggattcattct 8364 8383 1 3 SEQ ID NO: 852 gaactccccactggagctg 1004 1023 SEQ ID NO: 1855 cagctcaaccgtacagttc 11159 11176 1 3 SEQ ID NO: 853 atatcttcatgggagtcaa 1015 1035 SEQ ID NO: 1856 tgacttcagtgcagaatat 10329 10345 1 3 SEQ ID NO: 854 gtcatgctccccggaggca 1038 1057 SEQ ID NO: 1857 tggccccgtttaccatgac 10372 10391 1 3 SEQ ID NO: 855 gctgaagtttatcattcct 1042 1061 SEQ ID NO: 1858 aggaggctttaagttcagc 4868 4887 1 3 SEQ ID NO: 856 attccttccccaaagagac 1079 1098 SEQ ID NO: 1859 gtctcttcctccatggaat 5872 5891 1 3 SEQ ID NO: 857 ctcattgagaacaggcagt 1105 1124 SEQ ID NO: 1860 actgactgcacgctttgag 7104 7123 1 3 SEQ ID NO: 858 ttgagcagtattctgtcag 1106 1125 SEQ ID NO: 1861 cttagagaagtgtcttcaa 11044 11063 1 3 SEQ ID NO: 859 accttgtccagtgaagtcc 1122 1141 SEQ ID NO: 1862 ggacggtactgtcccaggt 12252 12271 1 3 SEQ ID NO: 860 ccagtgaagtccaaattcc 1146 1167 SEQ ID NO: 1863 ggaaggcagagtttactgg 6318 6337 1 3 SEQ ID NO: 861 acattcagaacaagaaaat 1168 1187 SEQ ID NO: 1864 atttcctaaagctggatgc 1359 1378 1 3 SEQ ID NO: 862 gaaaaatcaagggtgttat 1190 1209 SEQ ID NO: 1865 ataaactgcaagatttttc 7104 7123 1 3 SEQ ID NO: 863 aaatcaagggtgttatttc 1192 1211 SEQ ID NO: 1866 gaaacaatgcattagattt 12289 12308 1 3 SEQ ID NO: 864 tggcattatgatgaagaga 1204 1223 SEQ ID NO: 1867 tctcccgtgtataatgcca 13938 12957 1 3 SEQ ID NO: 865 aagagaagattgaatttga 1207 1228 SEQ ID NO: 1868 tcaaaacctactgtctcct 12271 12290 1 3 SEQ ID NO: 866 aaatgacttccaatttccc 1208 1227 SEQ ID NO: 1869 gggaactacaatttcattt 8520 8539 1 3 SEQ ID NO: 867 atgacttccaatttccctg 1223 1242 SEQ ID NO: 1870 caggctgattacgagtcat 4725 4744 1 3 SEQ ID NO: 868 acttccaatttccctgtgg 1269 1278 SEQ ID NO: 1871 ccacgaaaaatatggaagt 4993 5012 1 3 SEQ ID NO: 869 agttgcaatgagctcatgg 1288 1307 SEQ ID NO: 1872 ccatcagttcagataaact 7231 7250 1 3 SEQ ID NO: 870 tttgcaagaccacctcaat 1377 1396 SEQ ID NO: 1873 attgacctgtccattcaaa 12315 12334 1 3 SEQ ID NO: 871 gaaggagttcaacctccag 1380 1399 SEQ ID NO: 1874 ctggaattgtcattccttc 13043 13062 1 3 SEQ ID NO: 872 acttccacatcccagaaaa 1407 1426 SEQ ID NO: 1875 ttttaacaaaaagtggaag 5710 5729 1 3 SEQ ID NO: 873 ctcttcttaaaaagcgatg 1470 1480 SEQ ID NO: 1876 catcactgccaaaggagag 3928 3947 1 3 SEQ ID NO: 874 aaaagcgatggccgggtcc 1491 1510 SEQ ID NO: 1877 tgactcactcattgatttt 4652 4671 1 3 SEQ ID NO: 875 ttcctttgccttttggtgg 1492 1511 SEQ ID NO: 1878 ccacaaaacaatgaaggga 7856 7875 1 3 SEQ ID NO: 876 caagtctgtgggattccat 1497 1515 SEQ ID NO: 1879 atgggaaaaaacaggcttg 12279 12298 1 3 SEQ ID NO: 877 aagtccctacttttaccat 1557 1576 SEQ ID NO: 1880 atgggaagtataagaactt 6345 6364 1 3 SEQ ID NO: 878 tgcctctcctgggtgttct 1567 1586 SEQ ID NO: 1881 agaaaaacaaacaaacgga 7153 7172 1 3 SEQ ID NO: 879 accagcacagaccatttca 1574 1593 SEQ ID NO: 1882 tgaagtgtagtctcctggt 6428 6447 1 3 SEQ ID NO: 880 ccagcacagaccatttcag 1601 1620 SEQ ID NO: 1883 ctgaaatacaatgctctgg 9954 9973 1 3 SEQ ID NO: 881 actatcatgtgatgggtct 1607 1628 SEQ ID NO: 1884 agacacctgattttatagt 8392 8411 1 3 SEQ ID NO: 882 accacagatgtctgcttca 1617 1636 SEQ ID NO: 1885 tgaaggctgactctgtggt 11648 11665 1 3 SEQ ID NO: 883 ccacagagtgtctgcttcg 1619 1638 SEQ ID NO: 1886 ctgagcaacaatttgtgga 13912 1931 1 3 SEQ ID NO: 884 tttggactccaaaaagaaa 1655 1674 SEQ ID NO: 1887 tttctctcatgattacaaa 4421 4440 1 3 SEQ ID NO: 885 tcaaagaagtcaagattga 1664 1683 SEQ ID NO: 1888 tcaaggataactgtttgag 10875 10894 1 3 SEQ ID NO: 886 atgagaactacgagctgac 1665 1664 SEQ ID NO: 1889 gtcagatattgttgctcat 13991 14010 1 3 SEQ ID NO: 887 ttaaactctgacaccaatg 1677 1696 SEQ ID NO: 1890 cattcattgaagatgttaa 11165 11184 1 3 SEQ ID NO: 888 gaagtataagaactttgcc 1680 1699 SEQ ID NO: 1891 ggcaaatttgaaggacttc 5510 5529 1 3 SEQ ID NO: 889 aagtataagaaactttgca 1723 1742 SEQ ID NO: 1892 tggcaaatttgaaggactt 4534 4553 1 3 SEQ ID NO: 890 ttcttcagcctgctttctg 1726 1745 SEQ ID NO: 1893 cagaatccagatacaagaa 7234 7253 1 3 SEQ ID NO: 891 ctggatcactaaattccca 1729 1748 SEQ ID NO: 1894 tgggtctttccagagcccg 4282 4301 1 3 SEQ ID NO: 892 aaattaatagtggtgctca 1742 1761 SEQ ID NO: 1895 tgagaagccccaagaattt 2559 1578 1 3 SEQ ID NO: 893 ggccattccagaagggaag 1744 1763 SEQ ID NO: 1896 cttccgttctgtaatggcc 11299 11818 1 3 SEQ ID NO: 894 tgccatctcgagagttcca 1802 1821 SEQ ID NO: 1897 tggaactctctccatggca 9038 9057 1 3 SEQ ID NO: 895 catgtcaaacactttgtta 1816 1835 SEQ ID NO: 1898 taacaaattccttgacatg 7140 7159 1 3 SEQ ID NO: 896 tttgttataaatcttattg 1817 1836 SEQ ID NO: 1899 caataagatcaatagcaaa 7139 7158 1 3 SEQ ID NO: 897 tctggaaaaagggtcatga 1823 1842 SEQ ID NO: 1900 tccatgtcccatttacaga 6561 6600 1 3 SEQ ID NO: 898 cagctcttgttcaggtcca 1828 1847 SEQ ID NO: 1901 tggacctgcaccaaagctg 2752 2771 1 3 SEQ ID NO: 899 ggaggttccccagctctgc 1834 1853 SEQ ID NO: 1902 gcagccctgggaaaactcc 4534 4553 1 3 SEQ ID NO: 900 ctgttttgaagactctcca 1869 1888 SEQ ID NO: 1903 tggagggtagtcataacag 5326 5345 1 3 SEQ ID NO: 901 agtggctgaaacgtgtgca 1875 1894 SEQ ID NO: 1904 tgcagagctttctgccact 12669 12688 1 3 SEQ ID NO: 902 ccaaaatagaagggaatct 1879 1898 SEQ ID NO: 1905 agattcctttctttttcaa 8901 8920 1 3 SEQ ID NO: 903 tgaagagaagattgaattt 1884 1903 SEQ ID NO: 1906 aaattctcttttattttca 8990 9009 1 3 SEQ ID NO: 904 agtggtggcaacaccagca 1888 1907 SEQ ID NO: 1907 tgctagtgaggccaacact 7623 7642 1 3 SEQ ID NO: 905 aaggctccacaagtcatca 1892 1911 SEQ ID NO: 1908 tgatgatatctggaacctt 12737 12756 1 3 SEQ ID NO: 906 gtccgccaggtttctagca 1893 1912 SEQ ID NO: 1909 tgctaagaaccttactgac 12268 12287 1 3 SEQ ID NO: 907 tgatatctggaaccttgga 1500 1919 SEQ ID NO: 1910 ttcactgttcctgaaatca 9344 9363 1 3 SEQ ID NO: 908 gtcaagttgagcaatttct 1926 1945 SEQ ID NO: 1911 agaaaaggcacaccttgac 9392 9411 1 3 SEQ ID NO: 909 atccagatggaaaagggaa 1933 1952 SEQ ID NO: 1912 ttccaatttccctgtggat 1978 1997 1 3 SEQ ID NO: 910 atttgtttgtcaaagaagt 1941 1960 SEQ ID NO: 1913 acttcagagagaaatacat 7907 7926 1 3 SEQ ID NO: 911 ctggaaaatgtcagcctgg 1946 1967 SEQ ID NO: 1914 ccagacttcagttaccagc 1970 1989 1 3 SEQ ID NO: 912 accaggaggttcttcttca 1972 1991 SEQ ID NO: 1915 tgaagtgtgatctcctggt 12342 12361 1 3 SEQ ID NO: 913 aaagaagttctgaaaagat 1989 2008 SEQ ID NO: 1916 attccatcacaaaatcctt 7028 7047 1 3 SEQ ID NO: 914 gctacagcttatggctcca 2021 2040 SEQ ID NO: 1917 tggatctaaatgcagtagc 6970 6989 1 3 SEQ ID NO: 915 atcaatatgatcaatttgt 2023 2042 SEQ ID NO: 1918 caaagaaatcaagattgat 10276 10295 1 3 SEQ ID NO: 916 gaattatctttaaaacatt 2071 2090 SEQ ID NO: 1919 atgtggacaaatataccgg 5449 5468 1 3 SEQ ID NO: 917 cgaggcccgcgctgctggc 2075 2094 SEQ ID NO: 1920 gccagaagtgagatcctcg 4892 4911 1 3 SEQ ID NO: 918 acaactatgaggctgagag 2076 2095 SEQ ID NO: 1921 ctctgagcaacaaattttt 5279 5298 1 3 SEQ ID NO: 919 gctgagagttccagtggag 2093 2112 SEQ ID NO: 1922 ctccatggcaaatgtcagg 3453 3472 1 3 SEQ ID NO: 920 tgaagaaaaaccaagaact 2150 2169 SEQ ID NO: 1923 gagtcattgaggttcttca 10924 10943 1 3 SEQ ID NO: 921 cctacttacatcctgaaca 2154 2173 SEQ ID NO: 1924 tgttcataagggaggtagg 12257 12276 1 3 SEQ ID NO: 922 ctacttacatcctgaacat 2183 2202 SEQ ID NO: 1925 atgttcataagggaggtag 7842 7861 1 3 SEQ ID NO: 923 gagacagaagccaagagcc 2185 2204 SEQ ID NO: 1926 gcttggttttgcccagtct 4076 4097 1 3 SEQ ID NO: 924 cactcactttaccgtcaag 2196 2215 SEQ ID NO: 1927 cttgaacaccaaagtcact 12950 12969 1 3 SEQ ID NO: 925 ctgatcagcagcagccagt 2201 2220 SEQ ID NO: 1928 actgggaagtgcttatcag 13513 13532 1 3 SEQ ID NO: 926 actggacgctaagaggaag 2202 2221 SEQ ID NO: 1929 cttccccaaagagaggagt 13512 13531 1 3 SEQ ID NO: 927 agaggaagcatgtggcaga 2207 2226 SEQ ID NO: 1930 tctggcatttactttctct 4531 4550 1 3 SEQ ID NO: 928 tgaagactctccaggaact 2212 2231 SEQ ID NO: 1931 agttgaaggagactattca 3777 3795 1 3 SEQ ID NO: 929 ctctgagcaaaatatccag 2218 2237 SEQ ID NO: 1932 ctggttactgagctgagag 7790 7809 1 3 SEQ ID NO: 930 atgaagcagtcacatctct 2221 2240 SEQ ID NO: 1933 agagctgccagtcttcatt 6722 6741 1 3 SEQ ID NO: 931 ttgccacagctgattgagg 2239 2258 SEQ ID NO: 1934 cctcctacagtggtggcaa 6715 6734 1 3 SEQ ID NO: 932 agctgattgaggtgtccag 2323 2342 SEQ ID NO: 1935 ctggattccacatgcagct 2668 2687 1 3 SEQ ID NO: 933 tgctccactcacatcctcc 2329 2348 SEQ ID NO: 1936 ggaggctttaagttcagca 6272 8291 1 3 SEQ ID NO: 934 tgaaacgtgtgcatgtcaa 2349 2368 SEQ ID NO: 1937 ttgggagagacaagtttca 4254 4273 1 3 SEQ ID NO: 935 gacattgctaattacctga 2368 2377 SEQ ID NO: 1938 tcagaagctaagcaatgtc 9946 9985 1 3 SEQ ID NO: 936 ttcttcttcagactttcct 2369 2378 SEQ ID NO: 1939 aggagagtccaaatttaga 10801 10820 1 3 SEQ ID NO: 937 ccaatatcttgaaactcag 2362 2381 SEQ ID NO: 1940 tctgaattcattcaattgg 10359 13078 1 3 SEQ ID NO: 938 aaagttagtgaaaagaagt 2366 2385 SEQ ID NO: 1941 aactaccctcactgccttt 7973 7992 1 3 SEQ ID NO: 939 aagttagtgaaagaagttc 2377 2396 SEQ ID NO: 1942 gaacctctggcattttact 9534 9553 1 3 SEQ ID NO: 940 aaagaagttctgaaagaat 2376 2397 SEQ ID NO: 1943 attctctggtaactacttt 9533 9552 1 3 SEQ ID NO: 941 tttggctataccaaagatg 2379 2398 SEQ ID NO: 1944 catcttaggcactgacaaa 9532 9551 1 3 SEQ ID NO: 942 tgttgagaagctgattaaa 2393 2412 SEQ ID NO: 1945 tttagccatcggctcaaca 6866 6885 1 3 SEQ ID NO: 943 caggaagggctcaaagaat 2400 3419 SEQ ID NO: 1946 attcctttaacaattcctg 8240 8259 1 3 SEQ ID NO: 944 aggaagggctcaaagaatg 2401 2420 SEQ ID NO: 1947 cattcctttaacaattcct 8239 8258 1 3 SEQ ID NO: 945 gaagggctcaaagaatgac 2408 2427 SEQ ID NO: 1948 gtcagtcttcaggctcttc 3680 3899 1 3 SEQ ID NO: 946 caaagaatgacttttttct 2409 2428 SEQ ID NO: 1949 agaaggatggcattttttg 3679 3698 1 3 SEQ ID NO: 947 catggagaatgcctttgaa 2430 2449 SEQ ID NO: 1950 ttcagagccaaagtccatg 8563 8582 1 3 SEQ ID NO: 948 ggagccaaggctggagtaa 241 2450 SEQ ID NO: 1951 ttactccaacgccagctcc 8562 8581 1 3 SEQ ID NO: 949 tcattccttccccaaagag 2447 2466 SEQ ID NO: 1952 ctctctggggcatctatgc 12519 12536 1 3 SEQ ID NO: 950 acctatgagctccagagag 2458 2475 SEQ ID NO: 1953 ctctcaagaccacagaagg 2726 2745 1 3 SEQ ID NO: 951 gggcaaaaacgtcttacag 2461 2480 SEQ ID NO: 1954 tctgaaagacaacgtgccc 10763 10782 1 3 SEQ ID NO: 952 accctggaccttcagaaca 2471 2490 SEQ ID NO: 1955 tgttgctaaggttcagggt 3530 3549 1 3 SEQ ID NO: 953 atgggcgacctaagttgtg 2475 2494 SEQ ID NO: 1956 cacaaattagtttcaccat 2664 2683 1 3 SEQ ID NO: 954 gatgaagagaagattgaat 2493 2512 SEQ ID NO: 1957 atccagcttccccacactt 7931 7950 1 3 SEQ ID NO: 955 caatgtgataccaaaaaaa 2553 2572 SEQ ID NO: 1958 tttttggaaatgccattgt 3506 3525 1 3 SEQ ID NO: 956 gtagataccaaaaaaatga 2574 2593 SEQ ID NO: 1959 tcatgtgatgggtctctac 3827 3846 1 3 SEQ ID NO: 957 gcttcagttcatttggact 2582 2601 SEQ ID NO: 1960 agtcaagaaggacttaagc 6099 6118 1 3 SEQ ID NO: 958 tttgtttgtcaaagaagtc 2597 2618 SEQ ID NO: 1961 gacttcagagaaatacaaa 10710 10729 1 3 SEQ ID NO: 959 ttgtttgtcaaagaagtca 2600 2619 SEQ ID NO: 1962 tgacttcagagaaatacaa 12273 12292 1 3 SEQ ID NO: 960 tggcaatgggaaactcgct 2610 2629 SEQ ID NO: 1963 agcgagagtcccctgccat 5729 5748 1 3 SEQ ID NO: 961 aacctctggcatttacttt 2613 2632 SEQ ID NO: 1964 aaaggagatgtcaagggtt 5376 5394 1 3 SEQ ID NO: 962 catttactttctctcatga 2664 2703 SEQ ID NO: 1965 tcatttgaaagaataaatg 5072 5091 1 3 SEQ ID NO: 963 aaagtcagtgccctgctta 2690 2709 SEQ ID NO: 1966 taagaaccttactgacttt 3051 3070 1 3 SEQ ID NO: 964 tcccattttttgagacctt 2702 2721 SEQ ID NO: 1967 aaggacttcaggaatggga 4098 4117 1 3 SEQ ID NO: 965 catcaatattgatcaattt 2757 2776 SEQ ID NO: 1968 aaattaaaaagtcttgatg 11228 11247 1 3 SEQ ID NO: 966 taaagatagttatgattta 2759 2778 SEQ ID NO: 1969 taaaccaaaaacttggtta 4377 4396 1 3 SEQ ID NO: 967 tattgatgaaatcattgaa 2786 2605 SEQ ID NO: 1970 ttcaaagacttaaaaaata 12333 12352 1 3 SEQ ID NO: 968 atgatctacatttgtttat 2800 2819 SEQ ID NO: 1971 ataaagaaattaaagtcat 4171 4190 1 3 SEQ ID NO: 969 agagacacatacagaatat 2826 2845 SEQ ID NO: 1972 atatattgtcagtgcctct 12554 12573 1 3 SEQ ID NO: 970 gacacatacagaatataga 2833 2852 SEQ ID NO: 1973 tctaaattcagttcttgtc 4799 4818 1 3 SEQ ID NO: 971 agcatgtcaaacactttgt 2835 2554 SEQ ID NO: 1974 acaaagtcagtgccctgct 7631 7650 1 3 SEQ ID NO: 972 tttttagaggaaaccaagg 2861 2860 SEQ ID NO: 1975 ccttgtgtacaccaaaaac 7943 7952 1 3 SEQ ID NO: 973 ttttagaggaaaccaaggc 2871 2690 SEQ ID NO: 1976 gcctttgtgtacaccaaaa 6261 6230 1 3 SEQ ID NO: 974 ggaagatagacttcctgaa 2900 2919 SEQ ID NO: 1977 ttcagaaatactgtttttc 13206 13225 1 3 SEQ ID NO: 975 cactgtttctgagtcccag 2926 2945 SEQ ID NO: 1978 ctgggacctaccaagagtg 10183 10202 1 3 SEQ ID NO: 976 cacaaatcctttggctgtg 2931 2950 SEQ ID NO: 1979 cacatttcaaggaattgtg 11881 11900 1 3 SEQ ID NO: 977 ttcctggatacactgttcc 2932 2951 SEQ ID NO: 1980 ggaactgttgactcaggac 7098 7117 1 3 SEQ ID NO: 978 gaaatctcaagctttctct 2956 2975 SEQ ID NO: 1981 agagccaggtcgagctttc 3197 3216 1 3 SEQ ID NO: 979 tttcttcatcttcatctgt 2979 2998 SEQ ID NO: 1982 acagctgaaagagatgaaa 12023 12042 1 3 SEQ ID NO: 980 tctaccgctaaaggagcag 2961 3000 SEQ ID NO: 1983 ctgcacgctttgaggtaga 8333 8352 1 3 SEQ ID NO: 981 ctaccgctaaaggagcagt 3035 3054 SEQ ID NO: 1984 actgcacgctttgaggtcg 6432 6451 1 3 SEQ ID NO: 982 aggggcctcttttcaccaa 3066 3085 SEQ ID NO: 1985 ttggccaggaagtggccct 3184 3203 1 3 SEQ ID NO: 983 tctccatccctgtaaaaag 3093 3112 SEQ ID NO: 1986 ctttttcaccaacggagaa 12251 12270 1 3 SEQ ID NO: 984 gaaaaacaaagcagattat 3112 3131 SEQ ID NO: 1987 ataaactgcaagatttttc 5136 5155 1 3 SEQ ID NO: 985 actcactcattgattttct 3167 3186 SEQ ID NO: 1988 agaaaatcaggatcgtagt 6541 6560 1 3 SEQ ID NO: 986 taaactaatagatgtaatc 3194 3213 SEQ ID NO: 1989 gattaccaccagcagttta 13209 13226 1 3 SEQ ID NO: 987 caaaaacgagcttcggaag 3214 3233 SEQ ID NO: 1990 cttcgtgagaatatttgaa 9522 9541 1 3 SEQ ID NO: 988 tggaataatgctcagtgtt 3217 3236 SEQ ID NO: 1991 aacacttaacttgaattca 6947 6956 1 3 SEQ ID NO: 989 gatttgaaatccaaagaag 3225 3244 SEQ ID NO: 1992 cttcagagaaatacaaatc 4083 4102 1 3 SEQ ID NO: 990 atttgaaatccaaagaagt 3226 3245 SEQ ID NO: 1993 acttcagagaaatacaaat 4082 4101 1 3 SEQ ID NO: 991 atcaacagccgcttctttg 3280 3299 SEQ ID NO: 1994 caaagaagtcaagattgat 10543 10862 1 3 SEQ ID NO: 992 tgttttgaagactctccag 3261 3300 SEQ ID NO: 1995 ctggaaagttaaaacaaac 10842 10861 1 3 SEQ ID NO: 993 cccttctgatagatgtggt 3297 3316 SEQ ID NO: 1996 accaaagctggcaccaggg 10044 10063 1 3 SEQ ID NO: 994 tgagcaagtgaagaacttt 3363 3382 SEQ ID NO: 1997 aaagccattcagtctctca 4207 4226 1 3 SEQ ID NO: 995 atttgaaatccaaagaagt 3364 3383 SEQ ID NO: 1998 actttctaaacttgaaatt 4054 4073 1 3 SEQ ID NO: 996 atccaaagaagtcccggaa 3382 3401 SEQ ID NO: 1999 tccggggaaacctgggatt 9068 9087 1 3 SEQ ID NO: 997 agagcctacctccgcatct 3390 3409 SEQ ID NO: 2000 agatggtacgttagcctct 9218 9237 1 3 SEQ ID NO: 998 aatgcctttgaactcccca 3427 3446 SEQ ID NO: 2001 tgggaactacaattcattt 6627 6646 1 3 SEQ ID NO: 999 gaagtccaaattccggatt 3430 3449 SEQ ID NO: 2002 aatcttcaatttattcttc 6308 6327 1 3 SEQ ID NO: 1000 tgcaagcagaagccagaag 3441 3460 SEQ ID NO: 2003 cttcaggttccatcgtgca 13830 13849 1 3 SEQ ID NO: 1001 gaagagaagattgaatttg 3446 3465 SEQ ID NO: 2004 caaaacctactgtgcttcc 10212 10231 1 3 SEQ ID NO: 1002 atgctaaaggcacatatgg 3447 3486 SEQ ID NO: 2005 ccatatgaaagtcaagcat 10211 10230 1 3 SEQ ID NO: 1003 tccctcacctccacctctg 3465 3474 SEQ ID NO: 2006 cagattctcagatgaggga 11340 11359 1 3 SEQ ID NO: 2007 atttacagctctgacaagt 3947 3966 SEQ ID NO: 2313 acttttctaaacttgaaat 6335 6354 1 3 SEQ ID NO: 2008 aggagcctaccaaaataat 3963 3982 SEQ ID NO: 2314 attatgttgaaaacagtct 7356 7374 1 3 SEQ ID NO: 2009 aaagctgaagcacatcaat 3976 3995 SEQ ID NO: 2315 attgttgctcatctccttt 8302 8321 1 3 SEQ ID NO: 2010 ctgctggaaacaacgagaa 3967 4006 SEQ ID NO: 2316 ttctgattaccaccagcag 9039 9058 1 3 SEQ ID NO: 2011 ttgaaggaattcttgaaaa 3988 4007 SEQ ID NO: 2317 ttttaaaagaaatcttcaa 10446 10485 1 3 SEQ ID NO: 2012 gaagtaaaagaaaattttg 3990 4009 SEQ ID NO: 2318 caaaacctactgtctcttc 12022 12041 1 3 SEQ ID NO: 2013 tgaagaagatggcaaattt 4038 4057 SEQ ID NO: 2319 aaatgtcagctcttgttca 10537 10556 1 3 SEQ ID NO: 2014 aggatctgagttatttttc 4044 4063 SEQ ID NO: 2320 gcaagtcagcccagttcct 8065 8084 1 3 SEQ ID NO: 2015 gtgcccttctcggttgctg 4066 4085 SEQ ID NO: 2321 cagccattgacatgagcac 8805 8824 1 3 SEQ ID NO: 2016 ggcgcggcctgcgctgctg 4067 4086 SEQ ID NO: 2322 cagctccacagactccgcc 5720 5739 1 3 SEQ ID NO: 2017 ctgcgctgctgctgctgct 4094 4113 SEQ ID NO: 2323 agcagaaggtgcgaagcag 5322 5341 1 3 SEQ ID NO: 2018 gctggtggcgggcgccagg 4134 4153 SEQ ID NO: 2324 cctggattccacatgcagc 6246 5265 1 3 SEQ ID NO: 2019 aagaggaaatgctggaaaa 4148 4167 SEQ ID NO: 2325 tttttcttcactacatctt 8835 5854 1 3 SEQ ID NO: 2020 ctggaaaatgtcagcctgg 4170 4189 SEQ ID NO: 2326 ccagacttccacatcccag 4727 4746 1 3 SEQ ID NO: 2021 tggagtccctgggactgct 4184 4203 SEQ ID NO: 2327 agcatgcctagtttctcca 5580 5599 1 3 SEQ ID NO: 2022 ggagtccctgggactgctg 4167 4206 SEQ ID NO: 2328 cagcatgcctagtttctcc 11663 11682 1 3 SEQ ID NO: 2023 tgggactgctgattcaaga 4188 4207 SEQ ID NO: 2329 tcttccatcacttgaccca 11662 11681 1 3 SEQ ID NO: 2024 ctgctgattcaagaagtgc 4224 4243 SEQ ID NO: 2330 gcacaccttgacattgcag 13351 13370 1 3 SEQ ID NO: 2025 tgccaccaggatcaactgc 4272 4291 SEQ ID NO: 2331 gcaggctgaactggtggca 8603 8622 1 3 SEQ ID NO: 2026 gccaccaggatcaactgca 4283 4302 SEQ ID NO: 2332 tgcaggctgaactggtggc 13161 13160 1 3 SEQ ID NO: 2027 tgcaaggttgagcgtggag 4295 4314 SEQ ID NO: 2333 cctccatcctctgatctga 6957 6976 1 3 SEQ ID NO: 2028 caaggttgagctggaggtt 4304 4323 SEQ ID NO: 2334 aacccctacatgaagcttg 11080 11099 1 3 SEQ ID NO: 2029 ctctgcagcttcatcctga 4305 4324 SEQ ID NO: 2335 tcaggaagcttctcaagag 11079 11098 1 3 SEQ ID NO: 2030 cagcttcatcctgaagaac 4311 4330 SEQ ID NO: 2336 ggtcttgagttaaatgctg 8569 8588 1 3 SEQ ID NO: 2031 gcttcatcctgaagaccag 4344 4363 SEQ ID NO: 2337 ctggacgctaagaggaagc 9976 9995 1 3 SEQ ID NO: 2032 tcatcctgaagaccagcca 4345 4364 SEQ ID NO: 2338 tggcatggcattatgatga 9975 9994 1 3 SEQ ID NO: 2033 gaaaaccaagaactctgag 4355 4374 SEQ ID NO: 2339 ctcaaccttaatgattttc 6479 6498 1 3 SEQ ID NO: 2034 agaactctgaggagtttgc 4398 4417 SEQ ID NO: 2340 gcaagctatatcagtattc 8677 6698 1 3 SEQ ID NO: 2035 tctgaggagtttgctgcag 4404 4423 SEQ ID NO: 2341 ctgcagggatcccccagat 7094 7113 1 3 SEQ ID NO: 2036 tttgctgcagccatgtcca 4441 4460 SEQ ID NO: 2342 tggaagtgtcagtggcaaa 9206 9225 1 3 SEQ ID NO: 2037 caagaggggcatcatttct 4448 4467 SEQ ID NO: 2343 agaataaatgacgttcttg 11294 11313 1 3 SEQ ID NO: 2038 tcactttaccgtcaagacg 4454 4473 SEQ ID NO: 2344 cgtctacactatcatgtga 12223 12242 1 3 SEQ ID NO: 2039 tttaccgtcaagacgagga 4455 4474 SEQ ID NO: 2345 tccttgacatgttgataaa 12222 12241 1 3 SEQ ID NO: 2040 cactggacgctaagaggaa 4457 4476 SEQ ID NO: 2346 ttccagaaagcagccagtg 9272 9291 1 3 SEQ ID NO: 2041 aggaagcatgtggcagaag 4535 4554 SEQ ID NO: 2347 cttcatacacattaatcct 112061 11225 1 3 SEQ ID NO: 2042 caaggagcaacacctcttc 4543 4562 SEQ ID NO: 2348 gaagtagtactgcatgttg 8014 8033 1 3 SEQ ID NO: 2043 acagactttgaaacttgaa 4561 4580 SEQ ID NO: 2349 ttcaattcttcaatgctgt 13414 13433 1 3 SEQ ID NO: 2044 tgatgaagcagtcacatct 4576 4597 SEQ ID NO: 2350 agatttgaggattccatca 11987 12006 1 3 SEQ ID NO: 2045 agcagtcacatctctcttg 4580 4599 SEQ ID NO: 2351 caaggagaaactgactcgt 8885 8884 1 3 SEQ ID NO: 2046 ccagccccatcactttaca 4597 4616 SEQ ID NO: 2352 tgtagtctcctggtgctgg 9237 9256 1 3 SEQ ID NO: 2047 ctccactccatcctccagg 4606 4625 SEQ ID NO: 2353 ctggagcttagtaatggag 9364 9383 1 3 SEQ ID NO: 2048 catgccaacccccttctga 4659 4678 SEQ ID NO: 2354 tcagatgagggaacacatg 6182 6201 1 3 SEQ ID NO: 2049 gagagatcttcaacatggc 4660 4679 SEQ ID NO: 2355 gccaccctggaactctctc 9300 9319 1 3 SEQ ID NO: 2050 tcaacatggcgagggatca 4684 4703 SEQ ID NO: 2356 tgatcccacctctcattga 9368 9387 1 3 SEQ ID NO: 2051 ccaccttgtatgcgctgag 4722 4741 SEQ ID NO: 2357 ctcagggatctgaaggtgg 12283 12302 1 3 SEQ ID NO: 2052 gtcaacaactatcataaga 4754 4773 SEQ ID NO: 2358 tcttgagttaaatgctgac 12255 12274 1 3 SEQ ID NO: 2053 tggacattgctaattacct 4755 4774 SEQ ID NO: 2359 aggtatattcgaaagtcca 12254 12273 1 3 SEQ ID NO: 2054 ggacattgctaattacctg 4785 4804 SEQ ID NO: 2360 caggtatattcgaaagtcc 5969 5988 1 3 SEQ ID NO: 2055 ttctgcgggtcattggaaa 4796 4815 SEQ ID NO: 2361 tttcacatgccaaggagaa 6912 6931 1 3 SEQ ID NO: 2056 ccagaactcaagtcttcaa 4860 4879 SEQ ID NO: 2362 ttgaagtgtatgtctcctg 13024 13043 1 3 SEQ ID NO: 2057 agtcttcaatcctgaaatg 4899 4918 SEQ ID NO: 2363 catttctgattggtggact 6893 6912 1 3 SEQ ID NO: 2058 tgagcaagtgaagaacttt 4932 4951 SEQ ID NO: 2364 aaagtgccacttttactca 5340 5359 1 3 SEQ ID NO: 2059 agcaagtgaagaactttgt 4955 4974 SEQ ID NO: 2365 acaaagtcagtgccctgct 12541 12560 1 3 SEQ ID NO: 2060 tctgaaagaatctcaactt 4973 4992 SEQ ID NO: 2366 aagtccataatggttcaga 10095 10114 1 3 SEQ ID NO: 2061 actgtcatggacttcagaa 4999 5016 SEQ ID NO: 2367 ttctgaatatattgtcagt 14011 14030 1 3 SEQ ID NO: 2062 acttgacccagcctcagcc 5032 5051 SEQ ID NO: 2368 ggctcaccctgagagaagt 6786 6805 1 3 SEQ ID NO: 2063 tccaaataactaccttcct 5075 5094 SEQ ID NO: 2369 aggaagatatgaagatgga 6177 5196 1 3 SEQ ID NO: 2064 actaccctcactgcctttg 5084 5103 SEQ ID NO: 2370 caaatttgtggagggtagt 8234 6520 1 3 SEQ ID NO: 2065 ttggatttgcttcagctga 5108 5127 SEQ ID NO: 2371 tcagtataagtacaaccaa 9214 6233 1 3 SEQ ID NO: 2066 ttggaagctctttttggga 5127 5146 SEQ ID NO: 2372 tcccgattcacgcttccaa 6891 6910 1 3 SEQ ID NO: 2067 ggaagctctttttgggaag 5140 5159 SEQ ID NO: 2373 cttcagaaagctaccttcc 6491 6510 1 3 SEQ ID NO: 2068 tttttcccagacagtgtca 5142 5161 SEQ ID NO: 2374 tgaccttctctaagcaaaa 6428 6447 1 3 SEQ ID NO: 2069 agacagtgtcaacaaagct 5185 5204 SEQ ID NO: 2375 agcttggttttgccagtct 9498 7517 1 3 SEQ ID NO: 2070 ctttggctataccaaagat 5223 5242 SEQ ID NO: 2376 atctcgtgtctaggaaaag 8141 8160 1 3 SEQ ID NO: 2071 caaagatgataaacatgag 5239 5258 SEQ ID NO: 2377 ctcaaggataacgtgtttg 11348 11367 1 3 SEQ ID NO: 2072 gatatggtaaatggaataa 6295 5314 SEQ ID NO: 2378 ttatcttataattatatca 11969 11988 1 3 SEQ ID NO: 2073 ggaataatgctcagtgttg 5328 5347 SEQ ID NO: 2379 caaacacttacttgaattc 8478 6497 1 3 SEQ ID NO: 2074 tttgaaatccaaagaagtc 5341 5360 SEQ ID NO: 2380 gacttcagagaaatacaaa 5738 5757 1 3 SEQ ID NO: 2075 gatcccccagatgattgga 5345 5364 SEQ ID NO: 2381 tccaatttccctgtggatc 6694 6713 1 3 SEQ ID NO: 2076 cagatattggagaggtcaa 5378 5397 SEQ ID NO: 2382 tgaccacacaaaacagtct 10988 11007 1 3 SEQ ID NO: 2077 agaatgacttttttctctc 5381 5400 SEQ ID NO: 2383 tgaagtccggattcattct 12482 12501 1 3 SEQ ID NO: 2078 gaactccccactggagctg 5387 5406 SEQ ID NO: 2384 cagctcaaccgtacagttc 11844 11863 1 3 SEQ ID NO: 2079 atatcttcatgggagtcaa 5412 5431 SEQ ID NO: 2385 tgacttcagtgcagaatat 7362 7381 1 3 SEQ ID NO: 2080 gtcatgctccccggaggca 5427 5446 SEQ ID NO: 2386 tggccccgtttaccatgac 8014 8033 1 3 SEQ ID NO: 2081 gctgaagtttatcattcct 5435 5454 SEQ ID NO: 2387 aggaggctttaagttcagc 10666 10685 1 3 SEQ ID NO: 2082 attccttccccaaagagac 5480 5479 SEQ ID NO: 2388 gtctcttcctccatggaat 6570 5589 1 3 SEQ ID NO: 2083 ctcattgagaacaggcagt 5433 5502 SEQ ID NO: 2389 actgactgcacgctttgag 7267 7286 1 3 SEQ ID NO: 2084 ttgagcagtattctgtcag 5585 5607 SEQ ID NO: 2390 cttagagaagtgtcttcaa 9368 9387 1 3 SEQ ID NO: 2085 accttgtccagtgaagtcc 5591 5610 SEQ ID NO: 2391 ggacggtactgtcccaggt 6263 6262 1 3 SEQ ID NO: 2086 ccagtgaagtccaaattcc 5594 5513 SEQ ID NO: 2392 ggaaggcagagtttactgg 10119 10138 1 3 SEQ ID NO: 2087 acattcagaacaagaaaat 5608 5627 SEQ ID NO: 2393 atttcctaaagctggatgc 7057 7076 1 3 SEQ ID NO: 2088 gaaaaatcaagggtgttat 5618 5537 SEQ ID NO: 2394 ataaactgcaagatttttc 12150 12169 1 3 SEQ ID NO: 2089 aaatcaagggtgttatttc 5678 5597 SEQ ID NO: 2395 gaaacaatgcattagattt 9079 9098 1 3 SEQ ID NO: 2090 tggcattatgatgaagaga 5697 5716 SEQ ID NO: 2396 tctcccgtgtataatgcca 7442 7461 1 3 SEQ ID NO: 2091 aagagaagattgaatttga 5732 5751 SEQ ID NO: 2397 tcaaaacctactgtctcct 8588 8607 1 3 SEQ ID NO: 2092 aaatgacttccaatttccc 5782 5601 SEQ ID NO: 2398 gggaactacaatttcattt 8620 8639 1 3 SEQ ID NO: 2093 atgacttccaatttccctg 5763 5802 SEQ ID NO: 2399 caggctgattacgagtcat 8619 8638 1 3 SEQ ID NO: 2094 acttccaatttccctgtgg 5784 5803 SEQ ID NO: 2400 ccacgaaaaatatggaagt 8618 8367 1 3 SEQ ID NO: 2095 agttgcaatgagctcatgg 5786 5805 SEQ ID NO: 2401 ccatcagttcagataaact 12404 12423 1 3 SEQ ID NO: 2096 tttgcaagaccacctcaat 5792 5811 SEQ ID NO: 2402 attgacctgtccattcaaa 6493 6512 1 3 SEQ ID NO: 2097 gaaggagttcaacctccag 5793 5812 SEQ ID NO: 2403 ctggaattgtcattccttc 12967 12986 1 3 SEQ ID NO: 2098 acttccacatcccagaaaa 5851 5870 SEQ ID NO: 2404 ttttaacaaaaagtggaag 12205 12224 1 3 SEQ ID NO: 2099 ctcttcttaaaaagcgatg 5871 5890 SEQ ID NO: 2405 catcactgccaaaggagag 1080 1099 1 3 SEQ ID NO: 2100 aaaagcgatggccgggtcc 5906 5925 SEQ ID NO: 2406 tgactcactcattgatttt 8268 8285 1 3 SEQ ID NO: 2101 ttcctttgccttttggtgg 8975 6994 SEQ ID NO: 2407 ccacaaaacaatgaaggga 13604 13623 1 3 SEQ ID NO: 2102 caagtctgtgggattccat 5985 6004 SEQ ID NO: 2408 atgggaaaaaacaggcttg 13343 13362 1 3 SEQ ID NO: 2103 aagtccctacttttaccat 6001 6020 SEQ ID NO: 2409 atgggaagtataagaactt 6410 6429 1 3 SEQ ID NO: 2104 tgcctctcctgggtgttct 6038 6057 SEQ ID NO: 2410 agaaaaacaaacaaacgga 11602 11621 1 3 SEQ ID NO: 2105 accagcacagaccatttca 6053 6072 SEQ ID NO: 2411 tgaagtgtagtctcctggt 8029 8046 1 3 SEQ ID NO: 2106 ccagcacagaccatttcag 6076 6095 SEQ ID NO: 2412 ctgaaatacaatgctctgg 9674 9693 1 3 SEQ ID NO: 2107 actatcatgtgatgggtct 6095 6114 SEQ ID NO: 2413 agacacctgattttatagt 8591 8610 1 3 SEQ ID NO: 2108 accacagatgtctgcttca 6124 6143 SEQ ID NO: 2414 tgaaggctgactctgtggt 8265 8264 1 3 SEQ ID NO: 2109 ccacagagtgtctgcttcg 6190 6209 SEQ ID NO: 2415 ctgagcaacaatttgtgga 6980 6999 1 3 SEQ ID NO: 2110 tttggactccaaaaagaaa 6227 6246 SEQ ID NO: 2416 tttctctcatgattacaaa 9189 9188 1 3 SEQ ID NO: 2111 tcaaagaagtcaagattga 6249 6268 SEQ ID NO: 2417 tcaaggataactgtttgag 12962 12981 1 3 SEQ ID NO: 2112 atgagaactacgagctgac 6268 6267 SEQ ID NO: 2418 gtcagatattgttgctcat 101779 10198 1 3 SEQ ID NO: 2113 ttaaactctgacaccaatg 8275 6297 SEQ ID NO: 2419 cattcattgaagatgttaa 13647 13666 1 3 SEQ ID NO: 2114 gaagtataagaactttgcc 6280 6299 SEQ ID NO: 2420 ggcaaatttgaaggacttc 6350 6369 1 3 SEQ ID NO: 2115 aagtataagaaactttgca 6307 6326 SEQ ID NO: 2421 tggcaaatttgaaggactt 9568 9587 1 3 SEQ ID NO: 2116 ttcttcagcctgctttctg 6329 6348 SEQ ID NO: 2422 cagaatccagatacaagaa 9558 9577 1 3 SEQ ID NO: 2117 ctggatcactaaattccca 6336 6355 SEQ ID NO: 2423 tgggtctttccagagcccg 12940 12959 1 3 SEQ ID NO: 2118 aaattaatagtggtgctca 6415 6434 SEQ ID NO: 2424 tgagaagccccaagaattt 11821 11840 1 3 SEQ ID NO: 2119 ggccattccagaagggaag 6443 6462 SEQ ID NO: 2425 cttccgttctgtaatggcc 13614 13633 1 3 SEQ ID NO: 2120 tgccatctcgagagttcca 6462 6471 SEQ ID NO: 2426 tggaactctctccatggca 7716 7735 1 3 SEQ ID NO: 2121 catgtcaaacactttgtta 6461 6480 SEQ ID NO: 2427 taacaaattccttgacatg 12881 12880 1 3 SEQ ID NO: 2122 tttgttataaatcttattg 6469 6508 SEQ ID NO: 2428 caataagatcaatagcaaa 10471 10490 1 3 SEQ ID NO: 2123 tctggaaaaagggtcatga 6495 6514 SEQ ID NO: 2429 tccatgtcccatttacaga 11800 11819 1 3 SEQ ID NO: 2124 cagctcttgttcaggtcca 6526 6545 SEQ ID NO: 2430 tggacctgcaccaaagctg 11155 11174 1 3 SEQ ID NO: 2125 ggaggttccccagctctgc 6533 6552 SEQ ID NO: 2431 gcagccctgggaaaactcc 8372 8391 1 3 SEQ ID NO: 2126 ctgttttgaagactctcca 6536 6556 SEQ ID NO: 2432 tggagggtagtcataacag 9687 9706 1 3 SEQ ID NO: 2127 agtggctgaaacgtgtgca 6633 6652 SEQ ID NO: 2433 tgcagagctttctgccact 1925 1944 1 3 SEQ ID NO: 2128 ccaaaatagaagggaatct 6649 6668 SEQ ID NO: 2434 agattcctttctttttcaa 13807 13826 1 3 SEQ ID NO: 2129 tgaagagaagattgaattt 6675 6894 SEQ ID NO: 2435 aaattctcttttattttca 13120 13139 1 3 SEQ ID NO: 2130 agtggtggcaacaccagca 6689 6708 SEQ ID NO: 2436 tgctagtgaggccaacact 13856 13875 1 3 SEQ ID NO: 2131 aaggctccacaagtcatca 6690 6709 SEQ ID NO: 2437 tgatgatatctggaacctt 13855 13874 1 3 SEQ ID NO: 2132 gtccgccaggtttctagca 6695 6714 SEQ ID NO: 2438 tgctaagaaccttactgac 14076 14095 1 3 SEQ ID NO: 2133 tgatatctggaaccttgga 6711 6730 SEQ ID NO: 2439 ttcactgttcctgaaatca 8260 8279 1 3 SEQ ID NO: 2134 gtcaagttgagcaatttct 6739 6758 SEQ ID NO: 2440 agaaaaggcacaccttgac 12858 12875 1 3 SEQ ID NO: 2135 atccagatggaaaagggaa 6740 6759 SEQ ID NO: 2441 ttccaatttccctgtggat 8781 8780 1 3 SEQ ID NO: 2136 atttgtttgtcaaagaagt 6745 6764 SEQ ID NO: 2442 acttcagagagaaatacat 13093 13112 1 3 SEQ ID NO: 2137 ctggaaaatgtcagcctgg 6769 6788 SEQ ID NO: 2443 ccagacttcagttaccagc 11694 11713 1 3 SEQ ID NO: 2138 accaggaggttcttcttca 6772 6791 SEQ ID NO: 2444 tgaagtgtgatctcctggt 11802 11821 1 3 SEQ ID NO: 2139 aaagaagttctgaaaagat 6797 6816 SEQ ID NO: 2445 attccatcacaaaatcctt 11167 11186 1 3 SEQ ID NO: 2140 gctacagcttatggctcca 6816 6835 SEQ ID NO: 2446 tggatctaaatgcagtagc 9863 9882 1 3 SEQ ID NO: 2141 atcaatatgatcaatttgt 6820 6839 SEQ ID NO: 2447 caaagaaatcaagattgat 8005 8025 1 3 SEQ ID NO: 2142 gaattatctttaaaacatt 6880 6899 SEQ ID NO: 2448 atgtggacaaatataccgg 10080 10099 1 3 SEQ ID NO: 2143 cgaggcccgcgctgctggc 6886 6905 SEQ ID NO: 2449 gccagaagtgagatcctcg 9057 9076 1 3 SEQ ID NO: 2144 acaactatgaggctgagag 6916 6935 SEQ ID NO: 2450 ctctgagcaacaaattttt 9483 9502 1 3 SEQ ID NO: 2145 gctgagagttccagtggag 6942 6961 SEQ ID NO: 2451 ctccatggcaaatgtcagg 3680 3699 1 3 SEQ ID NO: 2146 tgaagaaaaaccaagaact 7052 7071 SEQ ID NO: 2452 gagtcattgaggttcttca 6183 6202 1 3 SEQ ID NO: 2147 cctacttacatcctgaaca 7053 7072 SEQ ID NO: 2453 tgttcataagggaggtagg 5326 5345 1 3 SEQ ID NO: 2148 ctacttacatcctgaacat 7062 7061 SEQ ID NO: 2454 atgttcataagggaggtag 13125 13144 1 3 SEQ ID NO: 2149 gagacagaagccaagagcc 7103 7122 SEQ ID NO: 2455 gcttggttttgcccagtct 12021 12040 1 3 SEQ ID NO: 2150 cactcactttaccgtcaag 7152 7171 SEQ ID NO: 2456 cttgaacaccaaagtcact 10323 10342 1 3 SEQ ID NO: 2151 ctgatcagcagcagccagt 7156 7176 SEQ ID NO: 2457 actgggaagtgcttatcag 12148 12167 1 3 SEQ ID NO: 2152 actggacgctaagaggaag 7215 7234 SEQ ID NO: 2458 cttccccaaagagaggagt 10160 10179 1 3 SEQ ID NO: 2153 agaggaagcatgtggcaga 7256 7275 SEQ ID NO: 2459 tctggcatttactttctct 11326 11345 1 3 SEQ ID NO: 2154 tgaagactctccaggaact 7283 7282 SEQ ID NO: 2460 agttgaaggagactattca 2069 2068 1 3 SEQ ID NO: 2155 ctctgagcaaaatatccag 7272 7291 SEQ ID NO: 2461 ctggttactgagctgagag 9061 9060 1 3 SEQ ID NO: 2156 atgaagcagtcacatctct 7280 7299 SEQ ID NO: 2462 agagctgccagtcttcatt 7435 7454 1 3 SEQ ID NO: 2157 ttgccacagctgattgagg 7284 7303 SEQ ID NO: 2463 cctcctacagtggtggcaa 10411 10430 1 3 SEQ ID NO: 2158 agctgattgaggtgtccag 7292 7311 SEQ ID NO: 2464 ctggattccacatgcagct 9945 9964 1 3 SEQ ID NO: 2159 tgctccactcacatcctcc 7345 7364 SEQ ID NO: 2465 ggaggctttaagttcagca 7414 7433 1 3 SEQ ID NO: 2160 tgaaacgtgtgcatgtcaa 7346 7355 SEQ ID NO: 2466 ttgggagagacaagtttca 7413 7432 1 3 SEQ ID NO: 2161 gacattgctaattacctga 7347 7366 SEQ ID NO: 2467 tcagaagctaagcaatgtc 10487 10506 1 3 SEQ ID NO: 2162 ttcttcttcagactttcct 7348 7367 SEQ ID NO: 2468 aggagagtccaaatttaga 10486 10505 1 3 SEQ ID NO: 2163 ccaatatcttgaaactcag 7349 7368 SEQ ID NO: 2469 tctgaattcattcaattgg 10485 10504 1 3 SEQ ID NO: 2164 aaagttagtgaaaagaagt 7372 7391 SEQ ID NO: 2470 aactaccctcactgccttt 11479 11498 1 3 SEQ ID NO: 2165 aagttagtgaaagaagttc 7398 7417 SEQ ID NO: 2471 gaacctctggcattttact 8783 8802 1 3 SEQ ID NO: 2166 aaagaagttctgaaagaat 7433 7452 SEQ ID NO: 2472 attctctggtaactacttt 7954 7983 1 3 SEQ ID NO: 2167 tttggctataccaaagatg 7434 7453 SEQ ID NO: 2473 catcttaggcactgacaaa 8630 8649 1 3 SEQ ID NO: 2168 tgttgagaagctgattaaa 7444 7463 SEQ ID NO: 2474 tttagccatcggctcaaca 10723 10742 1 3 SEQ ID NO: 2169 caggaagggctcaaagaat 7465 7464 SEQ ID NO: 2475 attcctttaacaattcctg 8401 8420 1 3 SEQ ID NO: 2170 aggaagggctcaaagaatg 7539 7558 SEQ ID NO: 2476 cattcctttaacaattcct 8532 8551 1 3 SEQ ID NO: 2171 gaagggctcaaagaatgac 7608 7627 SEQ ID NO: 2477 gtcagtcttcaggctcttc 10165 10184 1 3 SEQ ID NO: 2172 caaagaatgacttttttct 7633 7652 SEQ ID NO: 2478 agaaggatggcattttttg 10205 10224 1 3 SEQ ID NO: 2173 catggagaatgcctttgaa 7676 7695 SEQ ID NO: 2479 ttcagagccaaagtccatg 11013 11032 1 3 SEQ ID NO: 2174 ggagccaaggctggagtaa 7892 7711 SEQ ID NO: 2480 ttactccaacgccagctcc 6246 6266 1 3 SEQ ID NO: 2175 tcattccttccccaaagag 7714 7733 SEQ ID NO: 2481 ctctctggggcatctatgc 9369 9386 1 3 SEQ ID NO: 2176 acctatgagctccagagag 7715 7734 SEQ ID NO: 2482 ctctcaagaccacagaagg 8802 8821 1 3 SEQ ID NO: 2177 gggcaaaaacgtcttacag 7724 7743 SEQ ID NO: 2483 tctgaaagacaacgtgccc 10527 10546 1 3 SEQ ID NO: 2178 accctggaccttcagaaca 7730 7749 SEQ ID NO: 2484 tgttgctaaggttcagggt 11017 11036 1 3 SEQ ID NO: 2179 atgggcgacctaagttgtg 7734 7753 SEQ ID NO: 2485 cacaaattagtttcaccat 13673 13692 1 3 SEQ ID NO: 2180 gatgaagagaagattgaat 7745 7764 SEQ ID NO: 2486 atccagcttccccacactt 10275 10294 1 3 SEQ ID NO: 2181 caatgtgataccaaaaaaa 7762 7781 SEQ ID NO: 2487 tttttggaaatgccattgt 14018 14037 1 3 SEQ ID NO: 2182 gtagataccaaaaaaatga 7839 7858 SEQ ID NO: 2488 tcatgtgatgggtctctac 10200 10219 1 3 SEQ ID NO: 2183 gcttcagttcatttggact 7840 7859 SEQ ID NO: 2489 agtcaagaaggacttaagc 10199 10216 1 3 SEQ ID NO: 2184 tttgtttgtcaaagaagtc 7860 7879 SEQ ID NO: 2490 gacttcagagaaatacaaa 8951 8970 1 3 SEQ ID NO: 2185 ttgtttgtcaaagaagtca 7879 7898 SEQ ID NO: 2491 tgacttcagagaaatacaa 7965 7984 1 3 SEQ ID NO: 2186 tggcaatgggaaactcgct 7889 7908 SEQ ID NO: 2492 agcgagagtcccctgccat 73970 13989 1 3 SEQ ID NO: 2187 aacctctggcatttacttt 7921 7940 SEQ ID NO: 2493 aaaggagatgtcaagggtt 9580 8599 1 3 SEQ ID NO: 2188 catttactttctctcatga 7996 8015 SEQ ID NO: 2494 tcatttgaaagaataaatg 13175 13194 1 3 SEQ ID NO: 2189 aaagtcagtgccctgctta 8005 8024 SEQ ID NO: 2495 taagaaccttactgacttt 6821 6840 1 3 SEQ ID NO: 2190 tcccattttttgagacctt 8031 8080 SEQ ID NO: 2496 aaggacttcaggaatggga 9464 9483 1 3 SEQ ID NO: 2191 catcaatattgatcaattt 8055 8074 SEQ ID NO: 2497 aaattaaaaagtcttgatg 9881 9900 1 3 SEQ ID NO: 2192 taaagatagttatgattta 8081 8100 SEQ ID NO: 2498 taaaccaaaaacttggtta 10406 10425 1 3 SEQ ID NO: 2193 tattgatgaaatcattgaa 8137 8156 SEQ ID NO: 2499 ttcaaagacttaaaaaata 12924 12943 1 3 SEQ ID NO: 2194 atgatctacatttgtttat 8225 8244 SEQ ID NO: 2500 ataaagaaattaaagtcat 6514 8533 1 3 SEQ ID NO: 2195 agagacacatacagaatat 8312 8331 SEQ ID NO: 2501 atatattgtcagtgcctct 8876 8895 1 3 SEQ ID NO: 2196 gacacatacagaatataga 8331 8350 SEQ ID NO: 2502 tctaaattcagttcttgtc 8913 8932 1 3 SEQ ID NO: 2197 agcatgtcaaacactttgt 8379 8396 SEQ ID NO: 2503 acaaagtcagtgccctgct 11604 11623 1 3 SEQ ID NO: 2198 tttttagaggaaaccaagg 8391 8410 SEQ ID NO: 2504 ccttgtgtacaccaaaaac 11544 11583 1 3 SEQ ID NO: 2199 ttttagaggaaaccaaggc 8414 8433 SEQ ID NO: 2505 gcctttgtgtacaccaaaa 9730 9749 1 3 SEQ ID NO: 2200 ggaagatagacttcctgaa 8428 8447 SEQ ID NO: 2506 ttcagaaatactgtttttc 9662 9681 1 3 SEQ ID NO: 2201 cactgtttctgagtcccag 8500 8519 SEQ ID NO: 2507 ctgggacctaccaagagtg 13402 13421 1 3 SEQ ID NO: 2202 cacaaatcctttggctgtg 8501 8520 SEQ ID NO: 2508 cacatttcaaggaattgtg 13401 13420 1 3 SEQ ID NO: 2203 ttcctggatacactgttcc 8519 8538 SEQ ID NO: 2509 ggaactgttgactcaggac 11529 11548 1 3 SEQ ID NO: 2204 gaaatctcaagctttctct 8522 8541 SEQ ID NO: 2510 agagccaggtcgagctttc 13652 13671 1 3 SEQ ID NO: 2205 tttcttcatcttcatctgt 8541 8560 SEQ ID NO: 2511 acagctgaaagagatgaaa 12452 12471 1 3 SEQ ID NO: 2206 tctaccgctaaaggagcag 8596 8615 SEQ ID NO: 2512 ctgcacgctttgaggtaga 113801 11399 1 3 SEQ ID NO: 2207 ctaccgctaaaggagcagt 8608 8627 SEQ ID NO: 2513 actgcacgctttgaggtcg 13772 13791 1 3 SEQ ID NO: 2208 aggggcctcttttcaccaa 8670 8689 SEQ ID NO: 2514 ttggccaggaagtggccct 13414 1343 1 3 SEQ ID NO: 2209 tctccatccctgtaaaaag 8743 8762 SEQ ID NO: 2515 ctttttcaccaacggagaa 13372 13391 1 3 SEQ ID NO: 2210 gaaaaacaaagcagattat 8757 8776 SEQ ID NO: 2516 ataaactgcaagatttttc 12694 12713 1 3 SEQ ID NO: 2211 actcactcattgattttct 8821 8640 SEQ ID NO: 2517 agaaaatcaggatcgtagt 12251 12270 1 3 SEQ ID NO: 2212 taaactaatagatgtaatc 8921 8940 SEQ ID NO: 2518 gattaccaccagcagttta 10030 10049 1 3 SEQ ID NO: 2213 caaaaacgagcttcggaag 9052 9071 SEQ ID NO: 2519 cttcgtgagaatatttgaa 12610 12629 1 3 SEQ ID NO: 2214 tggaataatgctcagtgtt 9059 9078 SEQ ID NO: 2520 aacacttaacttgaattca 7300 7919 1 3 SEQ ID NO: 2215 gatttgaaatccaaagaag 9069 9088 SEQ ID NO: 2521 cttcagagaaatacaaatc 13558 13577 1 3 SEQ ID NO: 2216 atttgaaatccaaagaagt 9133 9152 SEQ ID NO: 2522 acttcagagaaatacaaat 12496 12517 1 3 SEQ ID NO: 2217 atcaacagccgcttctttg 9134 9153 SEQ ID NO: 2523 caaagaagtcaagattgat 2890 2908 1 3 SEQ ID NO: 2218 tgttttgaagactctccag 9213 9232 SEQ ID NO: 2524 ctggaaagttaaaacaaac 13630 13649 1 3 SEQ ID NO: 2219 cccttctgatagatgtggt 9222 9241 SEQ ID NO: 2525 accaaagctggcaccaggg 13486 13505 1 3 SEQ ID NO: 2220 tgagcaagtgaagaacttt 9275 9294 SEQ ID NO: 2526 aaagccattcagtctctca 10372 10391 1 3 SEQ ID NO: 2221 atttgaaatccaaagaagt 9304 9323 SEQ ID NO: 2527 actttctaaacttgaaatt 9840 9850 1 3 SEQ ID NO: 2222 atccaaagaagtcccggaa 9397 9416 SEQ ID NO: 2528 tccggggaaacctgggatt 14030 14048 1 3 SEQ ID NO: 2223 agagcctacctccgcatct 9427 9446 SEQ ID NO: 2529 agatggtacgttagcctct 13372 13391 1 3 SEQ ID NO: 2224 aatgcctttgaactcccca 9455 9474 SEQ ID NO: 2530 tgggaactacaattcattt 11726 11745 1 3 SEQ ID NO: 2225 gaagtccaaattccggatt 9470 9489 SEQ ID NO: 2531 aatcttcaatttattcttc 11711 11730 1 3 SEQ ID NO: 2226 tgcaagcagaagccagaag 9494 9513 SEQ ID NO: 2532 cttcaggttccatcgtgca 13198 13217 1 3 SEQ ID NO: 2227 gaagagaagattgaatttg 9497 9516 SEQ ID NO: 2533 caaaacctactgtgcttcc 9613 9632 1 3 SEQ ID NO: 2228 atgctaaaggcacatatgg 9526 9545 SEQ ID NO: 2534 ccatatgaaagtcaagcat 11203 11222 1 3 SEQ ID NO: 2229 tccctcacctccacctctg 9553 9572 SEQ ID NO: 2535 cagattctcagatgaggga 3926 3947 1 3 SEQ ID NO: 2230 atttacagctctgacaagt 9570 9589 SEQ ID NO: 2536 acttttctaaacttgaaat 13013 13032 1 3 SEQ ID NO: 2231 aggagcctaccaaaataat 9582 9601 SEQ ID NO: 2537 attatgttgaaaacagtct 10414 10433 1 3 SEQ ID NO: 2232 aaagctgaagcacatcaat 9583 9602 SEQ ID NO: 2538 attgttgctcatctccttt 11283 11302 1 3 SEQ ID NO: 2233 ctgctggaaacaacgagaa 9632 9651 SEQ ID NO: 2539 ttctgattaccaccagcag 9797 9816 1 3 SEQ ID NO: 2234 ttgaaggaattcttgaaaa 9712 9731 SEQ ID NO: 2540 ttttaaaagaaatcttcaa 11680 11699 1 3 SEQ ID NO: 2235 gaagtaaaagaaaattttg 9781 9800 SEQ ID NO: 2541 caaaacctactgtctcttc 12754 1273 1 3 SEQ ID NO: 2236 tgaagaagatggcaaattt 9851 9870 SEQ ID NO: 2542 aaatgtcagctcttgttca 8930 8949 1 3 SEQ ID NO: 2237 aggatctgagttatttttc 9863 9887 SEQ ID NO: 2543 gcaagtcagcccagttcct 13199 13218 1 3 SEQ ID NO: 2238 gtgcccttctcggttgctg 9886 9905 SEQ ID NO: 2544 cagccattgacatgagcac 4834 4853 1 3 SEQ ID NO: 2239 ggcgcggcctgcgctgctg 9942 9961 SEQ ID NO: 2545 cagctccacagactccgcc 11072 11091 1 3 SEQ ID NO: 2240 ctgcgctgctgctgctgct 10105 10124 SEQ ID NO: 2546 agcagaaggtgcgaagcag 6432 6451 1 3 SEQ ID NO: 2241 gctggtggcgggcgccagg 10159 10178 SEQ ID NO: 2547 cctggattccacatgcagc 7216 7235 1 3 SEQ ID NO: 2242 aagaggaaatgctggaaaa 10193 10212 SEQ ID NO: 2548 tttttcttcactacatctt 12881 12900 1 3 SEQ ID NO: 2243 ctggaaaatgtcagcctgg 10196 10215 SEQ ID NO: 2549 ccagacttccacatcccag 12423 12442 1 3 SEQ ID NO: 2244 tggagtccctgggactgct 10224 10243 SEQ ID NO: 2550 agcatgcctagtttctcca 13920 13939 1 3 SEQ ID NO: 2245 ggagtccctgggactgctg 10297 10316 SEQ ID NO: 2551 cagcatgcctagtttctcc 8187 8206 1 3 SEQ ID NO: 2246 tgggactgctgattcaaga 10322 10341 SEQ ID NO: 2552 tcttccatcacttgaccca 7153 7172 1 3 SEQ ID NO: 2247 ctgctgattcaagaagtgc 10369 10388 SEQ ID NO: 2553 gcacaccttgacattgcag 10770 10789 1 3 SEQ ID NO: 2248 tgccaccaggatcaactgc 10445 10454 SEQ ID NO: 2554 gcaggctgaactggtggca 13015 13034 1 3 SEQ ID NO: 2249 gccaccaggatcaactgca 10465 10474 SEQ ID NO: 2555 tgcaggctgaactggtggc 12851 12870 1 3 SEQ ID NO: 2250 tgcaaggttgagcgtggag 10468 10485 SEQ ID NO: 2556 cctccatcctctgatctga 10916 10935 1 3 SEQ ID NO: 2251 caaggttgagctggaggtt 10474 10493 SEQ ID NO: 2557 aacccctacatgaagcttg 7140 7159 1 3 SEQ ID NO: 2252 ctctgcagcttcatcctga 10504 10523 SEQ ID NO: 2558 tcaggaagcttctcaagag 12234 12253 1 3 SEQ ID NO: 2253 cagcttcatcctgaagaac 10540 10559 SEQ ID NO: 2559 ggtcttgagttaaatgctg 12025 12047 1 3 SEQ ID NO: 2254 gcttcatcctgaagaccag 10585 10584 SEQ ID NO: 2560 ctggacgctaagaggaagc 10641 10660 1 3 SEQ ID NO: 2255 tcatcctgaagaccagcca 10702 10721 SEQ ID NO: 2561 tggcatggcattatgatga 12281 12300 1 3 SEQ ID NO: 2256 gaaaaccaagaactctgag 10715 10734 SEQ ID NO: 2562 ctcaaccttaatgattttc 12737 12756 1 3 SEQ ID NO: 2257 agaactctgaggagtttgc 10852 10871 SEQ ID NO: 2563 gcaagctatatcagtattc 2459 2478 1 3 SEQ ID NO: 2258 tctgaggagtttgctgcag 10889 10908 SEQ ID NO: 2564 ctgcagggatcccccagat 12952 12971 1 3 SEQ ID NO: 2259 tttgctgcagccatgtcca 10890 10909 SEQ ID NO: 2565 tggaagtgtcagtggcaaa 12951 12970 1 3 SEQ ID NO: 2260 caagaggggcatcatttct 10906 10925 SEQ ID NO: 2566 agaataaatgacgttcttg 14058 14077 1 3 SEQ ID NO: 2261 tcactttaccgtcaagacg 10907 10926 SEQ ID NO: 2567 cgtctacactatcatgtga 14057 14076 1 3 SEQ ID NO: 2262 tttaccgtcaagacgagga 10932 10951 SEQ ID NO: 2568 tccttgacatgttgataaa 12248 12267 1 3 SEQ ID NO: 2263 cactggacgctaagaggaa 10979 10998 SEQ ID NO: 2569 ttccagaaagcagccagtg 11178 11197 1 3 SEQ ID NO: 2264 aggaagcatgtggcagaag 10986 11005 SEQ ID NO: 2570 cttcatacacattaatcct 13339 13358 1 3 SEQ ID NO: 2265 caaggagcaacacctcttc 10987 11006 SEQ ID NO: 2571 gaagtagtactgcatgttg 13338 13357 1 3 SEQ ID NO: 2266 acagactttgaaacttgaa 10995 11014 SEQ ID NO: 2572 ttcaattcttcaatgctgt 13814 13633 1 3 SEQ ID NO: 2267 tgatgaagcagtcacatct 11010 11029 SEQ ID NO: 2573 agatttgaggattccatca 9272 9291 1 3 SEQ ID NO: 2268 agcagtcacatctctcttg 11024 11043 SEQ ID NO: 2574 caaggagaaactgactcgt 13158 13177 1 3 SEQ ID NO: 2269 ccagccccatcactttaca 11071 11090 SEQ ID NO: 2575 tgtagtctcctggtgctgg 10458 10477 1 3 SEQ ID NO: 2270 ctccactccatcctccagg 11107 11126 SEQ ID NO: 2576 ctggagcttagtaatggag 7564 7583 1 3 SEQ ID NO: 2271 catgccaacccccttctga 11191 11210 SEQ ID NO: 2577 tcagatgagggaacacatg 12362 12381 1 3 SEQ ID NO: 2272 gagagatcttcaacatggc 11231 11250 SEQ ID NO: 2578 gccaccctggaactctctc 13140 13159 1 3 SEQ ID NO: 2273 tcaacatggcgagggatca 11269 11268 SEQ ID NO: 2579 tgatcccacctctcattga 8585 8904 1 3 SEQ ID NO: 2274 ccaccttgtatgcgctgag 11324 11343 SEQ ID NO: 2580 ctcagggatctgaaggtgg 13326 13345 1 3 SEQ ID NO: 2275 gtcaacaactatcataaga 11424 11443 SEQ ID NO: 2581 tcttgagttaaatgctgac 13102 13121 1 3 SEQ ID NO: 2276 tggacattgctaattacct 11445 11464 SEQ ID NO: 2582 aggtatattcgaaagtcca 12633 12652 1 3 SEQ ID NO: 2277 ggacattgctaattacctg 11528 11547 SEQ ID NO: 2583 caggtatattcgaaagtcc 8520 8539 1 3 SEQ ID NO: 2278 ttctgcgggtcattggaaa 11583 11602 SEQ ID NO: 2584 tttcacatgccaaggagaa 12685 12704 1 3 SEQ ID NO: 2279 ccagaactcaagtcttcaa 11600 11619 SEQ ID NO: 2585 ttgaagtgtatgtctcctg 11825 11844 1 3 SEQ ID NO: 2280 agtcttcaatcctgaaatg 11631 11650 SEQ ID NO: 2586 catttctgattggtggact 9223 9242 1 3 SEQ ID NO: 2281 tgagcaagtgaagaacttt 11683 11702 SEQ ID NO: 2587 aaagtgccacttttactca 12251 12270 1 3 SEQ ID NO: 2282 agcaagtgaagaactttgt 11684 11703 SEQ ID NO: 2588 acaaagtcagtgccctgct 12250 12269 1 3 SEQ ID NO: 2283 tctgaaagaatctcaactt 11695 11714 SEQ ID NO: 2589 aagtccataatggttcaga 13171 13190 1 3 SEQ ID NO: 2284 actgtcatggacttcagaa 11798 11818 SEQ ID NO: 2590 ttctgaatatattgtcagt 6496 6515 1 3 SEQ ID NO: 2285 acttgacccagcctcagcc 11995 12015 SEQ ID NO: 2591 ggctcaccctgagagaagt 11430 11449 1 3 SEQ ID NO: 2286 tccaaataactaccttcct 12048 12067 SEQ ID NO: 2592 aggaagatatgaagatgga 13278 13297 1 3 SEQ ID NO: 2287 actaccctcactgcctttg 12052 12071 SEQ ID NO: 2593 caaatttgtggagggtagt 13952 13971 1 3 SEQ ID NO: 2288 ttggatttgcttcagctga 12056 12085 SEQ ID NO: 2594 tcagtataagtacaaccaa 13703 13722 1 3 SEQ ID NO: 2289 ttggaagctctttttggga 12256 12275 SEQ ID NO: 2595 tcccgattcacgcttccaa 11659 11678 1 3 SEQ ID NO: 2290 ggaagctctttttgggaag 12292 12311 SEQ ID NO: 2596 cttcagaaagctaccttcc 3010 3029 1 3 SEQ ID NO: 2291 tttttcccagacagtgtca 12319 12338 SEQ ID NO: 2597 tgaccttctctaagcaaaa 8500 6519 1 3 SEQ ID NO: 2292 agacagtgtcaacaaagct 12354 12373 SEQ ID NO: 2597 agcttggttttgccagtct 12755 12774 1 3 SEQ ID NO: 2293 ctttggctataccaaagat 12457 12486 SEQ ID NO: 2598 atctcgtgtctaggaaaag 13107 13126 1 3 SEQ ID NO: 2294 caaagatgataaacatgag 12576 12595 SEQ ID NO: 2599 ctcaaggataacgtgtttg 12632 12651 1 3 SEQ ID NO: 2295 gatatggtaaatggaataa 12728 12747 SEQ ID NO: 2600 ttatcttataattatatca 12857 12875 1 3 SEQ ID NO: 2296 ggaataatgctcagtgttg 12869 12888 SEQ ID NO: 2601 caaacacttacttgaattc 13516 13535 1 3 SEQ ID NO: 2297 tttgaaatccaaagaagtc 12966 12985 SEQ ID NO: 2602 gacttcagagaaatacaaa 5794 5813 1 3 SEQ ID NO: 2298 gatcccccagatgattgga 12003 13012 SEQ ID NO: 2603 tccaatttccctgtggatc 13956 13975 1 3 SEQ ID NO: 2299 cagatattggagaggtcaa 13057 13076 SEQ ID NO: 2604 tgaccacacaaaacagtct 13192 13211 1 3 SEQ ID NO: 2300 agaatgacttttttctctc 13072 13091 SEQ ID NO: 2605 tgaagtccggattcattct 13807 13826 1 3 SEQ ID NO: 2301 gaactccccactggagctg 13142 13161 SEQ ID NO: 2606 cagctcaaccgtacagttc 9558 9577 1 3 SEQ ID NO: 2302 atatcttcatgggagtcaa 13172 13191 SEQ ID NO: 2607 tgacttcagtgcagaatat 11694 11713 1 3 SEQ ID NO: 2303 gtcatgctccccggaggca 13271 13290 SEQ ID NO: 2608 tggccccgtttaccatgac 13929 13948 1 3 SEQ ID NO: 2304 gctgaagtttatcattcct 13303 13322 SEQ ID NO: 2609 aggaggctttaagttcagc 8287 8306 1 3 SEQ ID NO: 2305 attccttccccaaagagac 13434 13453 SEQ ID NO: 2610 gtctcttcctccatggaat 13826 13845 1 3 SEQ ID NO: 2306 ctcattgagaacaggcagt 13501 13520 SEQ ID NO: 2611 actgactgcacgctttgag 13613 13832 1 3 SEQ ID NO: 2307 ttgagcagtattctgtcag 13562 13581 SEQ ID NO: 2612 cttagagaagtgtcttcaa 7991 8010 1 3 SEQ ID NO: 2308 accttgtccagtgaagtcc 13672 13691 SEQ ID NO: 2613 ggacggtactgtcccaggt 10414 10435 1 3 SEQ ID NO: 2309 ccagtgaagtccaaattcc 13685 13704 SEQ ID NO: 2614 ggaaggcagagtttactgg 7362 7381 1 3 SEQ ID NO: 2310 acattcagaacaagaaaat 13803 13822 SEQ ID NO: 2615 atttcctaaagctggatgc 10374 10393 1 3 SEQ ID NO: 2311 gaaaaatcaagggtgttat 14035 14054 SEQ ID NO: 2616 ataaactgcaagatttttc 7856 7875 1 3 SEQ ID NO: 2312 aaatcaagggtgttatttc 14049 14068 SEQ ID NO: 2617 gaaacaatgcattagattt 9834 9853 1 3 # = Match Number B = Middle Matching Bases

TABLE 9 Selected palindromic sequences from human ApoB Start End Start End Source Index Index Match Index Index # B SEQ ID NO: 2619 ggccattccagaagggaag 517 536 SEQ ID NO: 3948 cttccgttctgtaatggcc 5803 6322 1 9 SEQ ID NO: 2620 tgccatctcgagagttcca 4107 4126 SEQ ID NO: 3949 tggaactctctccatggca 10884 10903 1 8 SEQ ID NO: 2621 catgtcaaacactttgtta 7064 7083 SEQ ID NO: 3950 taacaaattccttgacatg 7368 7385 1 8 SEQ ID NO: 2622 ttgttataaatcttaattg 7076 7095 SEQ ID NO: 3951 caataagatcaatagcaaa 8999 9017 1 8 SEQ ID NO: 2623 tctggaaaagggtcatgga 8888 8907 SEQ ID NO: 3959 tccatgtcccatttacaga 11384 11383 1 8 SEQ ID NO: 2624 cagctcttgttcaggtcca 10908 10927 SEQ ID NO: 3960 tggacctgcaccaaagctg 13980 13979 1 8 SEQ ID NO: 2625 ggaggttccccagactcgc 354 363 SEQ ID NO: 3961 gcagccctgggaaaactcc 6465 5474 1 7 SEQ ID NO: 2626 ctgttttgaagactctcca 1088 1108 SEQ ID NO: 3962 tggagggtagtcataacag 10335 10354 1 7 SEQ ID NO: 2627 agtggctgaaacgtgtgca 1305 1324 SEQ ID NO: 3963 tgcagagctttctgcccac 13516 13635 1 7 SEQ ID NO: 2628 ccaaaatagaagggaatct 2076 2095 SEQ ID NO: 3964 agattcctttgccttttgg 4008 4027 1 7 SEQ ID NO: 2629 tgaagagaagattgaattt 3820 3647 SEQ ID NO: 3965 aaattctcttttcttttca 8220 9239 1 7 SEQ ID NO: 2630 agtggtgccaataccagca 4238 4257 SEQ ID NO: 3966 tgctagtgaggccaacact 10857 10676 1 7 SEQ ID NO: 2631 aaggctccacaagtcatca 5956 5977 SEQ ID NO: 3967 tgatgatatctggaacctt 10732 10751 1 7 SEQ ID NO: 2632 gtcagccaggtttatagca 7733 7752 SEQ ID NO: 3968 tgctaagaaccttactgac 7789 7808 1 7 SEQ ID NO: 2633 tgatatctggaaccttgaa 10735 10754 SEQ ID NO: 3969 ttcactgttcctgaaatca 7871 7890 1 7 SEQ ID NO: 2634 gtcaagttgagcaatttct 13431 13450 SEQ ID NO: 3970 agaaaaggcacaccttgac 11080 11099 1 7 SEQ ID NO: 2635 atccagatggaaaagggaa 13488 13507 SEQ ID NO: 3971 ttccccatttccctgtggt 3666 3707 1 7 SEQ ID NO: 2636 atttgtttgtcaaagaagt 4551 4570 SEQ ID NO: 3972 acttcagagaaatacaaat 11409 11427 4 6 SEQ ID NO: 2637 ctggaaaatgtcagcctgg 212 231 SEQ ID NO: 3973 ccagacttccgtttcccag 8243 6262 2 6 SEQ ID NO: 2638 accaggaggttcttcttca 1737 1756 SEQ ID NO: 3974 tgaagtgtagtctcctggt 5097 5118 2 6 SEQ ID NO: 2639 aaagaagttctgaaagaat 1964 1983 SEQ ID NO: 3975 attccatcacaatcctttt 9669 9688 2 6 SEQ ID NO: 2640 gctacagcttatggctcca 3578 3597 SEQ ID NO: 3976 tggatctaaatgcagtagc 11631 11650 2 6 SEQ ID NO: 2641 atccatcttgatcaatttg 6422 6441 SEQ ID NO: 3977 caaagaagtcaagattgat 4561 4580 2 6 SEQ ID NO: 2642 gaattatcttttaaaacat 7334 7353 SEQ ID NO: 3978 atgtgttaaccaaatattc 11502 11521 2 6 SEQ ID NO: 2643 cgaggcccgcgctgctggc 138 157 SEQ ID NO: 3979 gccagaagtgagatcctcg 3515 3534 1 6 SEQ ID NO: 2644 acaactatgaggctgagag 279 293 SEQ ID NO: 3980 ctctgagcaacaaatttgt 10317 10338 1 6 SEQ ID NO: 2645 gctgagagttccagtggcg 290 309 SEQ ID NO: 3981 ctccatggcaaatgtcagc 10593 10912 1 6 SEQ ID NO: 2646 tgaagaaaaccaagaactc 468 475 SEQ ID NO: 3982 gagtcattgcggttcttca 4937 4956 1 6 SEQ ID NO: 2647 cctcttacatcctgaacaa 506 586 SEQ ID NO: 3983 tgttcataagggaggtcgg 12774 12703 1 6 SEQ ID NO: 2648 ctacttacatcctgaacat 567 588 SEQ ID NO: 3984 atgttcataagggcggtag 12773 12782 1 6 SEQ ID NO: 2649 gagacagaagaagccaagc 623 642 SEQ ID NO: 3985 gcttggttttgccagtctc 2467 2488 1 6 SEQ ID NO: 2650 cactcactttaccgtcaag 679 698 SEQ ID NO: 3986 cttgaacacaaagtcagtg 8008 5027 1 6 SEQ ID NO: 2651 ctgatcagcagcagccagt 830 849 SEQ ID NO: 3987 actgggaagtgcttatcag 5245 5264 1 6 SEQ ID NO: 2652 actggccgctaagaggaag 862 961 SEQ ID NO: 3988 cttccccaaagagaccagt 2869 2917 1 6 SEQ ID NO: 2653 agaggaagcatgtggcaga 873 892 SEQ ID NO: 3989 tctggcatttactttctct 6929 5048 1 6 SEQ ID NO: 2654 tgaagactctccaggaact 1086 1114 SEQ ID NO: 3990 agttgaaggagactattca 7224 7243 1 6 SEQ ID NO: 2655 ctctgagcaaaatatccag 1129 1148 SEQ ID NO: 3991 ctggttactgagctgagag 1169 1188 1 6 SEQ ID NO: 2656 atgaagcagtcacatctct 1197 1216 SEQ ID NO: 3992 agagctgccagtccttcat 10024 10043 1 6 SEQ ID NO: 2657 ttgccacagctgattgagg 1217 1236 SEQ ID NO: 3993 cctcctacagtggtggcaa 4230 4249 1 6 SEQ ID NO: 2659 agctgattgaggtgtccag 1224 1243 SEQ ID NO: 3994 ctggattccacatgcagct 11856 11874 1 6 SEQ ID NO: 2650 tgctccactcacatcctcc 1286 1305 SEQ ID NO: 3995 ggaggctttaagttcagca 7609 7628 1 6 SEQ ID NO: 2660 tgaagcgtgtgcatgccaa 1311 1330 SEQ ID NO: 3996 ttgggagagacaagtttca 6508 6527 1 6 SEQ ID NO: 2661 gacattgctaattacctga 1511 1630 SEQ ID NO: 3997 tcagaagctaagcaatgtc 7240 7259 1 6 SEQ ID NO: 2662 ttcttcttcagactttcct 1746 1765 SEQ ID NO: 3998 aggagagtccaaattagaa 6506 8526 1 6 SEQ ID NO: 2663 ccaatatcttgaactcaga 1911 1930 SEQ ID NO: 3999 tctgaattcattcaattgg 6493 6512 1 6 SEQ ID NO: 2664 aaagttggtgaaagaagtt 1954 1973 SEQ ID NO: 4000 aactaccctcactgccttt 2140 2159 1 6 SEQ ID NO: 2665 aagtagtgaaagaaagttc 1955 1974 SEQ ID NO: 4001 gaacctctggcatttactt 5924 5943 1 6 SEQ ID NO: 2666 aaagaagttctgaaagaat 1964 1983 SEQ ID NO: 4002 attctctggtaactacttt 5490 5509 1 6 SEQ ID NO: 2667 tttggctataccaaagatg 2330 2349 SEQ ID NO: 4003 catcttaggcactgacaaa 5005 5024 1 6 SEQ ID NO: 2668 tgttgagaagctgattaaa 2389 2408 SEQ ID NO: 4004 tttagccatcggctcaaca 5708 5727 1 6 SEQ ID NO: 2669 caggaagggctcaaagaat 3569 2589 SEQ ID NO: 4005 attcctttaacaggttcag 9500 9519 1 6 SEQ ID NO: 2670 aggaagggctcaaagaatg 2570 2589 SEQ ID NO: 4006 cattcctttaacaattcct 9499 9518 1 6 SEQ ID NO: 2671 gaagggctcaaagaatgac 2572 2591 SEQ ID NO: 4007 gtcagtcttcaggctcttc 7922 7941 1 6 SEQ ID NO: 2672 caaagaatgacttttttct 2580 2599 SEQ ID NO: 4008 agaaggatggcattttttg 14008 14027 1 6 SEQ ID NO: 2673 catggagaatgcctttgaa 2611 2630 SEQ ID NO: 4009 ttcagagccaaagtccatg 7127 7146 1 6 SEQ ID NO: 2674 ggagccacggatggagtaa 2687 2706 SEQ ID NO: 4010 ttatccaacgccagctccc 3058 3077 1 6 SEQ ID NO: 2675 tcattccttccccaaagag 2892 2911 SEQ ID NO: 4011 ctctcggggcatctatgaa 5147 5166 1 6 SEQ ID NO: 2676 acctatgagctccagagag 3173 3192 SEQ ID NO: 4012 ctctcaagaccacagaggt 12984 13003 1 6 SEQ ID NO: 2677 gggcaaacgtcttacagaa 3373 3392 SEQ ID NO: 4013 tctgaaagaaacgtgtgca 12325 12344 1 6 SEQ ID NO: 2678 accctggacattcagaaca 3395 3414 SEQ ID NO: 4014 tgttgctaaggttcagggt 5683 5702 1 6 SEQ ID NO: 2679 atgggcgacctaagttgtg 3437 3456 SEQ ID NO: 4015 cacaaattagttcaccatt 8949 8968 1 6 SEQ ID NO: 2680 gatgaagagagagttgagt 3626 3645 SEQ ID NO: 4016 attccagcttccccacatc 8338 8357 1 6 SEQ ID NO: 2681 caatgtagataccaaaaaa 3664 3683 SEQ ID NO: 4017 tttttggaaatgccattgc 8651 6670 1 6 SEQ ID NO: 2682 gtagataccaaaaaaatga 3868 3687 SEQ ID NO: 4018 tcatgtgatgggtctcacc 4379 4398 1 6 SEQ ID NO: 2683 gcttcagttcatttggact 4517 4536 SEQ ID NO: 4019 agtcaagaaggacttaagc 5312 5331 1 6 SEQ ID NO: 2684 tttgtttgttcaaagaagt 4552 4571 SEQ ID NO: 4020 gactcagagaaatacaaac 11406 11427 1 6 SEQ ID NO: 2685 ttgtttgtcaaagaagtca 4553 4572 SEQ ID NO: 4021 tgactcagagaaatacaac 11407 11428 1 6 SEQ ID NO: 2686 tggcaatgggaaactcgct 5854 5873 SEQ ID NO: 4022 agcgagaatcaccctgcca 8227 8248 1 6 SEQ ID NO: 2687 aacctctggcatttacttt 5925 5944 SEQ ID NO: 4023 aaaggagatgtcaagggtt 10607 10626 1 6 SEQ ID NO: 2688 catttactttctctcatga 5934 5953 SEQ ID NO: 4024 tcatttgaaagaataaatg 7034 7053 1 6 SEQ ID NO: 2689 aaagtcagtgccctgctta 6017 6036 SEQ ID NO: 4025 taagaacctactgaacttt 7792 7811 1 6 SEQ ID NO: 2690 tcccattttttgagacctt 6330 6349 SEQ ID NO: 4026 aaggacttcaggaatggga 12012 12031 1 6 SEQ ID NO: 2691 catcaatattgatcaattt 6421 6440 SEQ ID NO: 4027 aatattaaaagtcttgatg 6740 5759 1 6 SEQ ID NO: 2692 taaagatagttatgattta 6673 6692 SEQ ID NO: 4028 taaaccaaaacttggttta 9027 9046 1 6 SEQ ID NO: 2693 tattgatgaaatcattgaa 6721 6740 SEQ ID NO: 4029 ttcaaagacttaaaacaat 8015 8034 1 6 SEQ ID NO: 2694 atgatctacatttgtttat 6798 6817 SEQ ID NO: 4030 ataaagaaattgaatcaat 7388 7407 1 6 SEQ ID NO: 2695 agagacacatacagaatat 6927 6946 SEQ ID NO: 4031 atatattgtcagtgcctct 13390 13409 1 6 SEQ ID NO: 2696 gacacatacagaatataga 6930 6949 SEQ ID NO: 4032 tctaaattcagttcttgtc 11335 11354 1 6 SEQ ID NO: 2697 agcatgtcaaacatttgtc 7062 7081 SEQ ID NO: 4033 acaaagtcagtgccctgct 6015 8034 1 6 SEQ ID NO: 2698 ttttagaggaaaccaaggc 7523 7542 SEQ ID NO: 4034 cctttgtgtacaccccaaa 11238 11257 1 6 SEQ ID NO: 2699 ttttagaggaaaccaaggc 7524 7543 SEQ ID NO: 4035 gcctttgtgtacaccaaaa 11237 11258 1 6 SEQ ID NO: 2700 ggaagatagacttcctgaa 9315 9334 SEQ ID NO: 4036 ttcagaaatactgtttccg 12832 12851 1 6 SEQ ID NO: 2701 cactgtttctgagtcccag 9342 9361 SEQ ID NO: 4037 ctgggacctaccaagagtg 12531 12550 1 6 SEQ ID NO: 2702 cacaaatcctttggctgtg 9676 9695 SEQ ID NO: 4038 cacatttcaggattgtggg 10071 10090 1 6 SEQ ID NO: 2703 ttcctggatacactgttcc 9801 9880 SEQ ID NO: 4039 ggaactgttgactcaggaa 12577 12596 1 6 SEQ ID NO: 2704 gaaatctcaagctttctat 10050 10069 SEQ ID NO: 4040 agagccaggtccgagcttc 11052 11071 1 6 SEQ ID NO: 2705 tttcttcatcttcatctgt 10218 10234 SEQ ID NO: 4041 acagctgaaagagatgaaa 13083 13082 1 6 SEQ ID NO: 2706 tctaccgctaaaggagcag 10529 10548 SEQ ID NO: 4042 ctgcacgcttgaggtagaa 11769 11788 1 6 SEQ ID NO: 2707 ctaccgtcaaaggagcagt 10530 10549 SEQ ID NO: 4043 actgcacgctttgaggtag 11768 11787 1 6 SEQ ID NO: 2708 agggcctctttttcaccaa 10839 10868 SEQ ID NO: 4044 ttggccaggaagtggccct 10965 10984 1 6 SEQ ID NO: 2709 ttctccatccctgtaaaag 11273 11292 SEQ ID NO: 4045 cttttcaccaaacgggaga 10846 10605 1 6 SEQ ID NO: 2710 gaaaaccaaagcagattct 11824 11343 SEQ ID NO: 4046 ataaactgcaagattttct 13608 13627 1 6 SEQ ID NO: 2711 actcactcattgatttcct 12690 12709 SEQ ID NO: 4047 agaaaatcaggatctgagt 14035 14054 1 6 SEQ ID NO: 2712 taaactaatagatgtaatc 12898 12917 SEQ ID NO: 4048 gattaccaccagcggttta 13586 13605 1 6 SEQ ID NO: 2713 caaaagagcttcaggaagc 13208 13227 SEQ ID NO: 4049 cttcgtgaagaatattttg 13268 13287 1 6 SEQ ID NO: 2714 tggaataatgctcagtgtt 2374 2393 SEQ ID NO: 4050 aacacttcattgaattcca 10670 10889 3 5 SEQ ID NO: 2715 gatttgaaatccaaaggag 2406 2427 SEQ ID NO: 4051 cttcagagaatacaaaatc 11410 11429 3 5 SEQ ID NO: 2716 atttgaaatccaaagaagt 2409 2428 SEQ ID NO: 4052 acttcagcgaaatacaaat 11409 11428 3 5 SEQ ID NO: 2717 atcaacagccgcttctttg 998 1017 SEQ ID NO: 4053 caaagaagtcaagattgat 4561 4580 2 5 SEQ ID NO: 2718 tgttttgaagactctccag 1090 1109 SEQ ID NO: 4054 ctggaaagttaaaacaaca 6963 6932 2 5 SEQ ID NO: 1719 cccttctgatagatgtggt 1332 1351 SEQ ID NO: 4055 accaaagctggcaccaggg 13989 13958 2 5 SEQ ID NO: 2720 tgagcaagtgaagaacttt 1876 1895 SEQ ID NO: 4056 aaagccattcagtctctca 12971 12990 2 5 SEQ ID NO: 2721 atttgaaatccaaagaagt 2409 2426 SEQ ID NO: 4057 acttttctaaacttgaaat 9063 9082 2 5 SEQ ID NO: 2722 atccaaagaagtcccggaa 2416 2435 SEQ ID NO: 4058 ttccggggaaacctgggat 12729 12748 2 5 SEQ ID NO: 2723 agagcctacctccgcatct 2438 2457 SEQ ID NO: 4059 agatggtacgttagcctct 11929 11948 2 5 SEQ ID NO: 2724 aatgcctttgaaactcccc 2615 2837 SEQ ID NO: 4060 tgggaactacaatttcatt 7020 7039 2 5 SEQ ID NO: 2725 gaagtccaaattccggatt 3305 3324 SEQ ID NO: 4061 aatcttcaatttattcttc 13823 13842 2 5 SEQ ID NO: 2726 tgcaagcagaagccagaag 3304 3625 SEQ ID NO: 4062 cttcaggtttccatcgtga 11384 11403 2 5 SEQ ID NO: 2727 gaagagaagattgaatttg 3629 3848 SEQ ID NO: 4063 caaaacctactgtctcctc 10467 10486 2 5 SEQ ID NO: 2728 atgctaaaggcacatatgg 4605 4524 SEQ ID NO: 4064 ccatatgaaagtcaagcat 12554 12683 2 5 SEQ ID NO: 2729 tccctcacctccacctctg 4745 4764 SEQ ID NO: 4065 cagattctcgatgagggat 8920 8939 2 5 SEQ ID NO: 2730 atttacagctctgacaagt 5435 5454 SEQ ID NO: 4066 acttttctaaacttgaatt 9353 9062 2 5 SEQ ID NO: 2731 aggagcctaccaaaattat 5602 5621 SEQ ID NO: 4067 attatgttgaaaacagttc 11838 11857 2 5 SEQ ID NO: 2732 aaagctgaagcacatcaat 8409 5428 SEQ ID NO: 4068 attgttgctcatctccttt 10202 10221 2 5 SEQ ID NO: 2733 ctgctggaaacaacgagaa 9426 9448 SEQ ID NO: 4069 tttctgataccacccacag 13682 13801 2 5 SEQ ID NO: 2734 ttgaaggaattcttgaaaa 9590 9509 SEQ ID NO: 4070 ttttaaaagaaaatctcaa 13813 12832 2 5 SEQ ID NO: 2735 gaagtaaaagaaatttggg 10751 10770 SEQ ID NO: 4071 caaaaacctactgtctggg 10467 10486 2 5 SEQ ID NO: 2736 tgaagaagatggcaaattt 11992 12011 SEQ ID NO: 4072 aaatgtcagctcttgttca 10902 10921 2 5 SEQ ID NO: 2737 aggatctgagttattttgc 14043 14062 SEQ ID NO: 4073 gcaagtcagcccagttcct 10928 10947 2 5 SEQ ID NO: 2738 gtgcccttctcggttgctg 26 45 SEQ ID NO: 4074 cagccattgacatgagcac 5748 5767 1 5 SEQ ID NO: 2739 ggcgctgcctgcgctgctg 154 173 SEQ ID NO: 4075 cagctccaccgactccgcc 3070 3089 1 5 SEQ ID NO: 2740 ctgcgctgctgctgctgct 162 181 SEQ ID NO: 4076 agcagaaggtgcaagcagg 3232 3251 1 5 SEQ ID NO: 2741 gctggtggcgggcgccagg 178 197 SEQ ID NO: 4077 cctggattccacatgcagc 11854 11673 1 5 SEQ ID NO: 2742 aagaggaaatgctggaaaa 201 220 SEQ ID NO: 4078 ttttcttcactactacttc 2592 2611 1 5 SEQ ID NO: 2743 ctggaaaatgtcagcctgg 212 231 SEQ ID NO: 4079 ccagacttccacatcccag 3923 3942 1 5 SEQ ID NO: 2744 tggagtccctgggactgct 305 323 SEQ ID NO: 4080 agcatgcctagtttctcca 9953 9972 1 5 SEQ ID NO: 2745 ggagctcccgggacttgtg 305 324 SEQ ID NO: 4081 cagtacgcctagtttctcc 9952 9971 1 5 SEQ ID NO: 2746 tgggactgctgattcaaga 313 332 SEQ ID NO: 4082 tcttccatcacttggaaac 2050 2039 1 5 SEQ ID NO: 2747 ctgctgattcaagaagtgc 316 337 SEQ ID NO: 4083 gcacaccttgacattgaca 11087 11106 1 5 SEQ ID NO: 2748 tgccaccaggatcaactgc 334 363 SEQ ID NO: 4084 gcaggctgaactggtggca 2725 2744 1 5 SEQ ID NO: 2749 gccaccaggatcaactgca 335 364 SEQ ID NO: 4085 tgcaggctgaactggttgc 2724 2743 1 5 SEQ ID NO: 2750 tgcaaggttgagctggagg 350 389 SEQ ID NO: 4086 cctccacctctgatctgca 4762 4771 1 5 SEQ ID NO: 2751 cataggttgagctggaggt 352 371 SEQ ID NO: 4089 aaccctacatggaaagctt 13783 13782 1 5 SEQ ID NO: 2752 ctctgcagtccatccctga 377 395 SEQ ID NO: 4090 tcaggaagcttctcaagag 13210 13238 1 5 SEQ ID NO: 2753 cagcttcatcctgaagacc 382 401 SEQ ID NO: 4091 ggtcttgagttaaatgctg 4985 5004 1 5 SEQ ID NO: 2754 gcttcatcctgaatgacag 384 403 SEQ ID NO: 4092 ctggacgctaagaggaagc 863 882 1 5 SEQ ID NO: 2755 tcatcctgaagaccagcca 387 406 SEQ ID NO: 4093 tggcatggcattatgatcc 3612 3631 1 5 SEQ ID NO: 2756 gaaaaccaagaactctgag 460 479 SEQ ID NO: 4094 ctcaaccttaatgattttc 8294 8313 1 5 SEQ ID NO: 2757 agaactctgaggagtttgc 468 437 SEQ ID NO: 4095 gcaagctctacagtattct 8385 8404 1 5 SEQ ID NO: 2758 tctgaggagtttgccgggt 473 492 SEQ ID NO: 4096 ctgcaggggatcccccaga 2534 2553 1 5 SEQ ID NO: 2759 tttgctgcagccatgtcca 482 801 SEQ ID NO: 4097 tggaagtgcagtggcaaat 10360 10399 1 5 SEQ ID NO: 2760 caagaggggcatcatttct 550 805 SEQ ID NO: 4098 agaataaatgacgttcttg 7043 7062 1 5 SEQ ID NO: 2761 tcactttaccgtcaagacg 662 701 SEQ ID NO: 4099 cgtctacactatagaaatt 4368 4387 1 5 SEQ ID NO: 2762 tttaccgtcaagacgaagg 686 705 SEQ ID NO: 4100 tccttgaccatgttgataa 7374 7393 1 5 SEQ ID NO: 2763 cactggacgctaagaaaga 861 880 SEQ ID NO: 4101 ttccagaaagcagccagtg 12506 12525 1 5 SEQ ID NO: 2764 aggaagcatgtggcagaag 675 894 SEQ ID NO: 4102 cttcatacacattatcctt 9996 10015 1 5 SEQ ID NO: 2765 caaggagcaacacctcttc 901 920 SEQ ID NO: 4103 gaagtagtactgcatcttg 6643 6882 1 5 SEQ ID NO: 2766 acagactttgaaacttgaa 957 988 SEQ ID NO: 4104 ttcaatcttcaatgctgtt 10508 10527 1 5 SEQ ID NO: 2767 tgatgaagcagtcacatct 1195 1214 SEQ ID NO: 4105 agatttgaggattccatca 7984 8003 1 5 SEQ ID NO: 2768 agcagtcacatctctcttg 1201 1220 SEQ ID NO: 4106 caaggagaaaactgactgc 6532 8551 1 5 SEQ ID NO: 2769 cagccccatcactttacat 1239 1258 SEQ ID NO: 4107 tgtagtctcctggtgctgg 5102 5121 1 5 SEQ ID NO: 2770 ctccactcatccttcagtg 1388 1307 SEQ ID NO: 4108 caggagcttagtaatggag 8717 8736 1 5 SEQ ID NO: 2771 catgcccaccccctcctgg 1322 1341 SEQ ID NO: 4109 tcagatgagggaacacatg 8927 8946 1 5 SEQ ID NO: 2772 gagagatcttcaacatggc 1398 1417 SEQ ID NO: 4110 gccaccctggaaactctct 10877 10896 1 5 SEQ ID NO: 2773 tcaaacatggcgacgcatc 1407 1428 SEQ ID NO: 4111 tgatcccacctctcattga 2973 2992 1 5 SEQ ID NO: 2774 ccaccttgtatgcgctgag 1437 1456 SEQ ID NO: 4112 ctcagggatctgaggggtg 8195 8214 1 5 SEQ ID NO: 2775 gtcaaacaactatatcata 1463 1482 SEQ ID NO: 4113 tcttgagttaaatgctgac 4987 5006 1 5 SEQ ID NO: 2776 tggacattgctaattacct 1509 1525 SEQ ID NO: 4114 aggtatattcgaaagtcca 12807 12826 1 5 SEQ ID NO: 2777 ggacattgctaattacctg 1510 1629 SEQ ID NO: 4115 caggtatattcgaaagtcc 12806 12825 1 5 SEQ ID NO: 2778 ttctgcgggtcattggaaa 1551 1600 SEQ ID NO: 4116 tttcacactgcccaagaaa 5522 6541 1 5 SEQ ID NO: 2779 ccagaactcaagtcttcaa 1628 1647 SEQ ID NO: 4117 ttgaagtgtgtctcctggc 5096 5115 1 5 SEQ ID NO: 2780 agtcttcaattcctgaagt 1638 1657 SEQ ID NO: 4118 catttctgattggtggact 7765 7784 1 5 SEQ ID NO: 2781 tgagcaagtgaagaaactt 1876 1895 SEQ ID NO: 4119 aaagtgccactttttacta 6191 6210 1 5 SEQ ID NO: 2782 agcaagtgaaagaacttgt 1878 1897 SEQ ID NO: 4120 acaaagtcagttgcctggt 6015 6034 1 5 SEQ ID NO: 2783 tctgaaaagaatctcaact 1972 1991 SEQ ID NO: 4121 aagtccataatggttcaga 12819 12833 1 5 SEQ ID NO: 2784 actgtcatggacttcagaa 1994 2013 SEQ ID NO: 4122 ttctgaatatattgtcagt 13384 13703 1 5 SEQ ID NO: 2785 acttgacccagcctcagcc 2059 2078 SEQ ID NO: 4123 ggctccccctgagagaagt 12399 1241 1 5 SEQ ID NO: 2786 tccaaataactaccttcct 2104 2123 SEQ ID NO: 4124 aggaagatagaagatggat 4720 4739 1 5 SEQ ID NO: 2787 actaccctcactgcctttg 2141 2160 SEQ ID NO: 4125 caaatttgtggagggtagt 10327 10346 1 5 SEQ ID NO: 2788 ttggatttgcttcagctga 2159 2176 SEQ ID NO: 4126 tcagtataaatacaccaaa 9400 9419 1 5 SEQ ID NO: 2789 ttggaagctcttgggaaat 2219 2238 SEQ ID NO: 4127 tcccgattcacgcttccaa 11585 11604 1 5 SEQ ID NO: 2790 ggaagctctttttgggaag 2221 2240 SEQ ID NO: 4128 cttcagaaagctaccttcc 7837 7955 1 5 SEQ ID NO: 2791 tttttcccagacagtgtca 2246 2285 SEQ ID NO: 4129 tgacctctctaagccaaat 4884 4903 1 5 SEQ ID NO: 2792 agacagtgtcaacaagctt 2254 2273 SEQ ID NO: 4130 agctggttttgcccagttt 2466 2405 1 5 SEQ ID NO: 2793 cttggctataccaaagatt 2329 2348 SEQ ID NO: 4131 atctcgtgtctaggaaaag 2976 5995 1 5 SEQ ID NO: 2794 caaagatgataaaacatgg 2341 2300 SEQ ID NO: 4132 ctcaaggataacgtgtttg 12617 12636 1 5 SEQ ID NO: 2795 gatatggtaaatggaataa 2363 2382 SEQ ID NO: 4133 tttacttagggttaatttg 13087 13106 1 5 SEQ ID NO: 2796 ggaataatgctcagtgttg 2375 2394 SEQ ID NO: 4134 caacacttacttgaattcc 10689 10688 1 5 SEQ ID NO: 2797 tttgaaatccaaagaaatc 2410 2429 SEQ ID NO: 4135 gacttcagagaaatacaat 11408 11427 1 5 SEQ ID NO: 2798 cagatgattggagaggtca 2542 2591 SEQ ID NO: 4136 tccaatttcctgtgggtgg 3689 3708 1 5 SEQ ID NO: 2799 agaatgacttttttcttca 2549 2588 SEQ ID NO: 4137 tgaccacacaaacagtgct 5371 6390 1 5 SEQ ID NO: 2800 agaatgactttttcttcac 2583 2505 SEQ ID NO: 4138 tgaagtccggcttctttct 11023 11042 1 5 SEQ ID NO: 2801 gaactcccactggagctgc 2627 2846 SEQ ID NO: 4139 cagctcaaccgtacagttc 11809 11856 1 5 SEQ ID NO: 2802 atatctaaggtggtccaca 2660 2879 SEQ ID NO: 4140 tgacttcagtgcagaatat 11974 11993 1 5 SEQ ID NO: 2803 gctgaagtttatcattcct 2675 2694 SEQ ID NO: 4141 tggccccgtttaccataga 5817 5836 1 5 SEQ ID NO: 2804 attccttcccccaaagaga 2881 2900 SEQ ID NO: 4142 aggaggctttaagttcagc 7608 627 1 5 SEQ ID NO: 2805 ctcattgagaaacaggcag 2894 2913 SEQ ID NO: 4143 gtctcttcctccatggaat 10478 10497 1 5 SEQ ID NO: 2806 ctcattgagaacaggcagt 2984 3003 SEQ ID NO: 4144 actgactgcacgctttgag 11764 11783 1 5 SEQ ID NO: 2807 ttgagcagtattctgtcag 3150 3169 SEQ ID NO: 4145 gctgagaaagtgcttcctt 12407 142428 1 5 SEQ ID NO: 2808 accttgtccagtgaagtcc 3293 3312 SEQ ID NO: 4146 ggacggtactgtcccaggt 12792 12811 1 5 SEQ ID NO: 2809 cacgtgaagtccaaattcc 3300 3319 SEQ ID NO: 4147 ggaaggcagagtttactgg 9150 9176 1 5 SEQ ID NO: 2810 acattcagaaaagaaagcg 3402 3421 SEQ ID NO: 4148 atttcctaaagctggattg 11175 11194 1 5 SEQ ID NO: 2811 gaaaaatcaagggtgttat 3471 3490 SEQ ID NO: 4149 ataaactgcaaagattttt 13609 13627 1 5 SEQ ID NO: 2812 aaatcaagggtgttatttc 3474 3493 SEQ ID NO: 4150 gaaaacaatgcattagatt 9753 9772 1 5 SEQ ID NO: 2813 tggcattatgatgaagaga 3817 3636 SEQ ID NO: 4151 tctcccgtgtatatgccaa 11789 11808 1 5 SEQ ID NO: 2814 aagagaagattgaatttga 3630 3649 SEQ ID NO: 4152 tcaaaacctactgtctctt 10466 10435 1 5 SEQ ID NO: 2815 aaatgacttccaatttccc 3681 3700 SEQ ID NO: 4153 gggaactacaatttcattt 7021 7040 1 5 SEQ ID NO: 2816 atgacttccaattttccct 3383 3702 SEQ ID NO: 4154 caggctgattacgagtcat 4925 4944 1 5 SEQ ID NO: 2817 acttccaattttccctgtg 3686 3705 SEQ ID NO: 4155 ccacgaaaaatatggaagt 10388 10387 1 5 SEQ ID NO: 2818 agttgcaatgagctcatgg 3811 3830 SEQ ID NO: 4156 ccatcagttcagataaact 7997 8016 1 5 SEQ ID NO: 2819 tttgcaagaccacctcaat 3868 3887 SEQ ID NO: 4157 attgacctgtccattcaaa 13679 136696 1 5 SEQ ID NO: 2820 gaaggagttcaacctccag 2892 3911 SEQ ID NO: 4158 ctggaaatttgtcattccc 11735 11756 1 5 SEQ ID NO: 2821 acttccacatcccagaaaa 3927 3946 SEQ ID NO: 4159 ttttaacaaaaagtggtgg 6629 6848 1 5 SEQ ID NO: 2822 ctcttcttaaaaagcgatg 3947 3966 SEQ ID NO: 4160 catcactgccaaaggagag 8494 8513 1 5 SEQ ID NO: 2823 aaaagagagggcgggtcaa 3956 3975 SEQ ID NO: 4161 tgactactcagggaaattt 12555 12707 1 5 SEQ ID NO: 2824 ttcctttgccttttggtgg 4011 4030 SEQ ID NO: 4162 ccacaaacaatgaagggaa 9264 9283 1 5 SEQ ID NO: 2825 caagtctcggggattccat 4087 4106 SEQ ID NO: 4163 atggaaaaaaaacagggct 9574 9693 1 5 SEQ ID NO: 2826 aagtccctactttttacat 4125 4144 SEQ ID NO: 4164 atgggaagtataagaactt 4842 4891 1 5 SEQ ID NO: 2827 tgcctctcctgggtgtttc 4157 4186 SEQ ID NO: 4165 agaaaacaaccaaggcaaa 9651 9670 1 5 SEQ ID NO: 2828 accagcacagaccacccca 4250 4269 SEQ ID NO: 4166 tgaagtgtagtctcctggt 5097 5118 1 5 SEQ ID NO: 2829 ccagcacagaccatttcag 4251 4270 SEQ ID NO: 4167 ctgaaatacaagcttctgg 6519 5538 1 5 SEQ ID NO: 2830 actatcatgtgatgggtct 4375 4394 SEQ ID NO: 4168 agacacctgatttttatat 7956 7976 1 5 SEQ ID NO: 2831 accacagatgtctgcttca 4304 4523 SEQ ID NO: 4169 tgaaggctgatttcttgtt 4290 4309 1 5 SEQ ID NO: 2832 ccacagatgtctgcttcag 4505 4524 SEQ ID NO: 4170 ctgagcaactttttttgtt 10319 10338 1 5 SEQ ID NO: 2833 tttggactccaaaaaagaa 4528 4547 SEQ ID NO: 4171 tttctctcatgatacaaaa 5941 5960 1 5 SEQ ID NO: 2834 tcaaagaagtcaagattga 4560 4579 SEQ ID NO: 4172 tcaaggataacgtgtttga 12618 12637 1 5 SEQ ID NO: 2835 atgagaactacgagctgac 4808 4625 SEQ ID NO: 4173 gtcagatattggtgctcat 10196 10214 1 5 SEQ ID NO: 2836 ttaaatctgacaacattgg 4826 4845 SEQ ID NO: 4174 cattcatttgaaggggaac 7350 7369 1 5 SEQ ID NO: 2837 gaagtataagaactttgcc 4846 4665 SEQ ID NO: 4175 ggcaaatttgaaggacttc 12002 12021 1 5 SEQ ID NO: 2838 aagtataagaactttgcca 4847 4866 SEQ ID NO: 4176 tggcaaatttgaaggactt 12001 12020 1 5 SEQ ID NO: 2839 ttcttcagcctgtttctgg 4949 4968 SEQ ID NO: 4177 cagaatccagatacaagaa 6892 6914 1 5 SEQ ID NO: 2840 ctggatcactaaattccca 4965 4984 SEQ ID NO: 4178 tgggtcttccagaaggccg 11041 11060 1 5 SEQ ID NO: 2841 aaattaatagtggtgctca 5022 5041 SEQ ID NO: 4179 tgagaagccccagaatttt 6266 6275 1 5 SEQ ID NO: 2842 agtgcaacgaccaacttga 5081 5100 SEQ ID NO: 4180 tcaaattcctggattacac 9866 9875 1 5 SEQ ID NO: 2843 ctgggaagtgcttatcagg 5246 5265 SEQ ID NO: 4181 cctgaccttcacataccag 8316 8337 1 5 SEQ ID NO: 2844 gcaaaaacaaatttcaggg 5286 5305 SEQ ID NO: 4182 aagtcaaagaaattttgct 10752 10771 1 5 SEQ ID NO: 2845 aaaaaacatttcaacttca 5288 5307 SEQ ID NO: 4183 tgaagtaaaagaaattttt 10750 10760 1 5 SEQ ID NO: 2846 tcagtcaagaagaacttaa 5310 5329 SEQ ID NO: 4184 ttaaggaacttccattcta 13371 13380 1 5 SEQ ID NO: 2847 tcaaatgacatgatgggct 5333 5352 SEQ ID NO: 4185 agcccatcaatatattgaa 6213 6232 1 5 SEQ ID NO: 2848 cacaaaacagtctgaacaa 5375 5394 SEQ ID NO: 4186 tgttttcaactgccttgta 11227 11246 1 5 SEQ ID NO: 2849 tcttcaaaacttgaccaca 5417 5436 SEQ ID NO: 4187 tgttttcctattttcaaga 12843 12862 1 5 SEQ ID NO: 2850 caagtttttattaggcact 5449 6468 SEQ ID NO: 4188 agttatttgctaaagcttg 14061 14070 1 5 SEQ ID NO: 2851 tggtaactactttaccagg 5496 5515 SEQ ID NO: 4189 ctgttttagaggaaagccg 7520 7539 1 5 SEQ ID NO: 2852 aacagtgacctgaaataca 5510 5529 SEQ ID NO: 4190 tgtatagcaaattcctgtt 5898 5917 1 5 SEQ ID NO: 2853 gggaaactacggctagaac 5552 5571 SEQ ID NO: 4191 gttccttccagatttccgc 10941 10960 1 5 SEQ ID NO: 2854 aacaaacttgccatcctcc 5028 5647 SEQ ID NO: 4192 gagaacagctttcgtttgg 11212 11231 1 5 SEQ ID NO: 2855 tcagcaagctataaagcag 5660 5679 SEQ ID NO: 4193 ctgctaagaaccttactga 7788 7807 1 5 SEQ ID NO: 2856 gcagacactgttgctaagg 5675 5694 SEQ ID NO: 4194 cctttcaagcactgactgc 11764 11773 1 5 SEQ ID NO: 2857 tctggggagaacatactgg 5674 5893 SEQ ID NO: 4195 ccaggttttccacaaccga 8046 8084 1 5 SEQ ID NO: 2858 ttctctcatgattacaaag 5942 5961 SEQ ID NO: 4196 ctttttcaaccaacgggaa 10846 10885 1 5 SEQ ID NO: 2859 ctgagcagacaggcacctg 6042 6661 SEQ ID NO: 4197 caggaggctttaagttgtc 7607 7526 1 5 SEQ ID NO: 2860 caatttaaacaaacaagat 6074 6093 SEQ ID NO: 4198 attccttcctttacaattg 8090 8109 1 5 SEQ ID NO: 2861 tggacgaaactctggctac 6146 6167 SEQ ID NO: 4199 gtcagcccaggttcctcaa 10932 10951 1 5 SEQ ID NO: 2862 ctttttactcagtgaccca 6200 6219 SEQ ID NO: 4200 tgggctaaacgtttaattg 7835 7854 1 5 SEQ ID NO: 2863 tcattgtggctttagaaga 6226 6244 SEQ ID NO: 4201 atcttcataagttcaatga 13182 13201 1 5 SEQ ID NO: 2864 aaaaccaagatgttcactc 6303 6322 SEQ ID NO: 4202 gagtgaaatgctgtttttt 8638 8867 1 5 SEQ ID NO: 2865 aggaatcgacaaccattat 6365 6384 SEQ ID NO: 4203 taatgatttcaatgttcct 6302 8321 1 5 SEQ ID NO: 2866 tagttgtactggaaaaacg 6384 6403 SEQ ID NO: 4204 acgttagcctctatagact 11935 11955 1 5 SEQ ID NO: 2867 ggaaaactgatcagagaag 6394 6413 SEQ ID NO: 4205 cttttacaattcatttccc 13022 13041 1 5 SEQ ID NO: 2868 gaaaacgtacaggaccccc 9695 6414 SEQ ID NO: 4206 gctttctctttcacaaatg 10080 10079 1 5 SEQ ID NO: 2869 aaagctgaagcccatcaat 6409 6425 SEQ ID NO: 4207 atttgattagttggcaagg 6992 7011 1 5 SEQ ID NO: 2870 aagtctaagacctaggaga 6410 6429 SEQ ID NO: 4208 tattgatgttagagtgctt 6991 7010 1 5 SEQ ID NO: 2871 tgaagcacatcaatattga 6414 6433 SEQ ID NO: 4209 tcaaccttaatgattttaa 8295 6314 1 5 SEQ ID NO: 2872 atcaatattgatcaatttg 6422 6441 SEQ ID NO: 4210 caaagccatacatgatggg 1658 1687 1 5 SEQ ID NO: 2873 taatgattatctgaattca 6484 8503 SEQ ID NO: 4211 tgaaatcattgaaaattta 6727 5746 1 5 SEQ ID NO: 2874 gattatcgaattcattcat 6488 6507 SEQ ID NO: 4212 tgaaagtacgctgaagttc 7102 7121 1 5 SEQ ID NO: 2875 aattgggagagaagccttt 6506 6625 SEQ ID NO: 4213 aaacattcctttaccactt 9496 9515 1 5 SEQ ID NO: 2876 aaaatagctattgctaata 3701 6720 SEQ ID NO: 4214 tattgaaatatgatttttt 6814 6839 1 5 SEQ ID NO: 2877 aaaattaacaagtccgtat 6739 6758 SEQ ID NO: 4215 atcataggttgaggttttt 6785 6784 1 5 SEQ ID NO: 2878 ttgaaaaatatttgattta 6818 6835 SEQ ID NO: 4216 ttaatctttcataagtcaa 13176 13198 1 5 SEQ ID NO: 2879 agacatccagcacctagct 6946 6965 SEQ ID NO: 4217 tgggctaaacgtttaattg 2468 2485 1 5 SEQ ID NO: 2880 caattcatttgaaagaaat 7029 7048 SEQ ID NO: 4218 tattgaaatatgatttttt 8090 8109 1 5 SEQ ID NO: 2881 aggtttaaatggataattt 7182 7201 SEQ ID NO: 4219 aattgttgaaagaaaaact 13155 13174 1 5 SEQ ID NO: 2882 cagaagctaagcaatgtcc 7241 7250 SEQ ID NO: 4220 ggacaaggcccagaatctg 12553 12572 1 5 SEQ ID NO: 2883 taagattaaatatttactt 7270 7289 SEQ ID NO: 4221 aaagaaacctatggcctta 13183 13182 1 5 SEQ ID NO: 2884 aaagattactttgagaaat 7277 7298 SEQ ID NO: 4222 attcttaacgggattcctt 9489 9508 1 5 SEQ ID NO: 2885 aggaaatagttggattacc 7280 7308 SEQ ID NO: 4223 taaagccattcaggtctct 12970 12969 1 5 SEQ ID NO: 2886 attttgatgatgctggccc 7303 7322 SEQ ID NO: 4224 gacatgtttgaagggtgga 7372 7398 1 5 SEQ ID NO: 2887 gaattatcttttaaacatt 734 7353 SEQ ID NO: 4225 tgggctaaacgtttaattg 7685 7704 1 5 SEQ ID NO: 2888 ttaccaccagtttgaagtt 7411 7430 SEQ ID NO: 4226 tattgaaatatgatttttt 10739 10758 1 5 SEQ ID NO: 2889 ttgcagtgtatcggacaga 7548 7567 SEQ ID NO: 4227 agcttgtttgccagtgggc 8420 8439 1 5 SEQ ID NO: 2890 cattcagaaccagggaaga 7699 7718 SEQ ID NO: 4228 tgaaatcattgaaaattta 12009 12020 1 5 SEQ ID NO: 2891 acaaactgtttatagtccc 7958 7977 SEQ ID NO: 4229 caggaggctttaagttgtc 12614 12633 1 5 SEQ ID NO: 2892 ggatttcaatacatttcac 7992 8011 SEQ ID NO: 4230 agcttgtttgccagtgggc 13124 13143 1 5 SEQ ID NO: 2893 ttgtagaaatgaaagtaaa 8112 8131 SEQ ID NO: 4231 taaagccattcaggtctct 12360 12379 1 5 SEQ ID NO: 2894 ctgaacagtgagctgcagt 8166 8175 SEQ ID NO: 4232 aattgttgaaagaaaaact 8809 8828 1 5 SEQ ID NO: 2895 aatccaatctcctctttgc 8407 3420 SEQ ID NO: 4233 taaagccattcaggtctct 11017 11036 1 5 SEQ ID NO: 2896 atttgattttcaagcaaag 8532 8554 SEQ ID NO: 4234 ttaatctttcataagtcaa 14023 14042 1 5 SEQ ID NO: 2897 ttttgattttcaagcaaat 8535 8552 SEQ ID NO: 4235 ggacaaggcccagaatctg 9622 9641 1 5 SEQ ID NO: 2898 tgatttcaagcaaatgcag 8536 8555 SEQ ID NO: 4236 attcttaacgggattcctt 14025 14044 1 5 SEQ ID NO: 2899 atgctgtttgggaaattgg 8645 8664 SEQ ID NO: 4237 aattgttgaaagaaaaact 11203 11222 1 5 SEQ ID NO: 2900 tgctgttttttggaaatgc 8646 8855 SEQ ID NO: 4238 agcttgtttgccagtgggc 11202 11221 1 5 SEQ ID NO: 2901 aaaaaaatacactggacgt 8706 8725 SEQ ID NO: 4239 ttaatctttcataagtcaa 10533 10652 1 5 SEQ ID NO: 2902 actggagcttagtaagggc 8716 8735 SEQ ID NO: 4240 agcttgtttgccagtgggc 1289 1308 1 5 SEQ ID NO: 2903 cttctggaaagagggtcat 8886 8905 SEQ ID NO: 4241 agcttgtttgccagtgggc 13759 13778 1 5 SEQ ID NO: 2904 ggaaaagggtcatggaaat 8891 8910 SEQ ID NO: 4242 attccttccttacaattgg 8282 9304 1 5 SEQ ID NO: 2905 gggcctgccccagatttct 8910 8929 SEQ ID NO: 4243 gagaacatttatggaggcc 9440 9450 1 5 SEQ ID NO: 2906 ttctcagataggggaacac 8924 8943 SEQ ID NO: 4244 gtgtcctcaagctgagggg 12416 12435 1 5 SEQ ID NO: 2907 gatgaaagaattaagggga 8930 8949 SEQ ID NO: 4245 attccagcttcccaactac 8338 8357 1 5 SEQ ID NO: 2908 cttggactgtcaaataagt 8996 9005 SEQ ID NO: 4246 cttatgggatttcctaagg 11167 11186 1 5 SEQ ID NO: 2909 gcatccacaaacaatggag 9200 9279 SEQ ID NO: 4247 cttcatcgttcatttgagt 10227 10246 1 5 SEQ ID NO: 2910 cacaaacaatgaagggaat 9265 8284 SEQ ID NO: 4248 cttatgggatttcctaagg 11456 11507 1 5 SEQ ID NO: 2911 ccaaaatttctctcggctt 9415 9434 SEQ ID NO: 4249 cttcatcgttcatttgagt 9671 9690 1 5 SEQ ID NO: 2912 caaattttctctgctaaaa 9416 9435 SEQ ID NO: 4250 ttccatcacaaatcctttg 9670 9689 1 5 SEQ ID NO: 2913 tctgctggaaacaacaacg 9425 9444 SEQ ID NO: 4251 tctcaaagagttacaacag 13328 13248 1 5 SEQ ID NO: 2914 cagaagctaagcaatgtcc 9426 6445 SEQ ID NO: 4252 tattgaaatatgatttttt 13228 13247 1 5 SEQ ID NO: 2915 taagattaaatatttactt 9441 9460 SEQ ID NO: 4253 aattgttgaaagaaaaact 8909 8928 1 5 SEQ ID NO: 2916 aaagattactttgagaaat 9475 9494 SEQ ID NO: 4254 ggacaaggcccagaatctg 13821 13840 1 5 SEQ ID NO: 2917 aggaaatagttggattacc 9565 9584 SEQ ID NO: 4255 aaagaaacctatggcctta 14021 14040 1 5 SEQ ID NO: 2918 attttgatgatgctggccc 9712 9731 SEQ ID NO: 4256 attcttaacgggattcctt 14033 14052 1 5 SEQ ID NO: 2919 gaattatcttttaaacatt 9751 9770 SEQ ID NO: 4257 taaagccattcaggtctct 5633 8662 1 5 SEQ ID NO: 2920 ttaccaccagtttgaagtt 10001 10020 SEQ ID NO: 4258 gacatgtttgaagggtgga 14089 14108 1 5 SEQ ID NO: 2921 ttgcagtgtatcggacaga 10194 10213 SEQ ID NO: 4259 tgggctaaacgtttaattg 4807 4826 1 5 SEQ ID NO: 2922 cattcagaaccagggaaga 10338 10355 SEQ ID NO: 4260 tattgaaatatgatttttt 27347 2753 1 5 SEQ ID NO: 2923 acaaactgtttatagtccc 10404 10423 SEQ ID NO: 4261 agcttgtttgccagtgggc 8368 8385 1 5 SEQ ID NO: 2924 ggatttcaatacatttcac 10405 10424 SEQ ID NO: 4262 tgaaatcattgaaaattta 8365 8384 1 5 SEQ ID NO: 2925 ttgtagaaatgaaagtaaa 10438 10455 SEQ ID NO: 4263 caggaggctttaagttgtc 13258 13277 1 5 SEQ ID NO: 2926 ttaccaccagtttgaagtt 10578 10597 SEQ ID NO: 4264 agcttgtttgccagtgggc 12974 12966 1 5 SEQ ID NO: 2927 ttgcagtgtatcggacaga 10863 10882 SEQ ID NO: 4265 taaagccattcaggtctct 12676 12695 1 5 SEQ ID NO: 2928 cattcagaaccagggaaga 10672 10691 SEQ ID NO: 4266 aattgttgaaagaaaaact 5008 6027 1 5 SEQ ID NO: 2929 acaaactgtttatagtccc 10751 10770 SEQ ID NO: 4267 taaagccattcaggtctct 5287 5306 1 5 SEQ ID NO: 2930 ggatttcaatacatttcac 10882 10901 SEQ ID NO: 4268 ttaatctttcataagtcaa 11372 11391 1 5 SEQ ID NO: 2931 ttgtagaaatgaaagtaaa 11164 11203 SEQ ID NO: 4269 ggacaaggcccagaatctg 11856 11874 1 5 SEQ ID NO: 2932 ctgaacagtgagctgcagt 11485 11504 SEQ ID NO: 4270 attcttaacgggattcctt 6705 5784 1 5 SEQ ID NO: 2933 aatccaatctcctctttgc 11613 11632 SEQ ID NO: 4271 aattgttgaaagaaaaact 12424 12443 1 5 SEQ ID NO: 2934 atttgattttcaagcaaag 11614 11633 SEQ ID NO: 4272 agcttgtttgccagtgggc 12423 12442 1 5 SEQ ID NO: 2935 ttttgattttcaagcaaat 11642 11861 SEQ ID NO: 4273 ttaatctttcataagtcaa 13103 13122 1 5 SEQ ID NO: 2936 acaaactgtttatagtccc 12229 12248 SEQ ID NO: 4274 agcttgtttgccagtgggc 13631 13650 1 5 SEQ ID NO: 2937 ggatttcaatacatttcac 12936 12957 SEQ ID NO: 4275 agcttgtttgccagtgggc 13826 13845 1 5 SEQ ID NO: 2938 ttgtagaaatgaaagtaaa 13034 13053 SEQ ID NO: 4276 attccttccttacaattgg 5014 8033 1 5 SEQ ID NO: 2939 ttaccaccagtttgaagtt 13097 13116 SEQ ID NO: 4277 tgggctaaacgtttaattg 10479 10498 1 5 SEQ ID NO: 2940 ttgcagtgtatcggacaga 13218 13237 SEQ ID NO: 4278 tattgaaatatgatttttt 13163 13202 1 5 SEQ ID NO: 2941 cattcagaaccagggaaga 13437 13450 SEQ ID NO: 4279 agcttgtttgccagtgggc 12932 12047 1 5 SEQ ID NO: 2942 acaaactgtttatagtccc 13712 13731 SEQ ID NO: 4280 tgaaatcattgaaaattta 7465 7484 1 5 SEQ ID NO: 2943 ggatttcaatacatttcac 13719 13738 SEQ ID NO: 4281 caggaggctttaagttgtc 7230 7258 1 5 SEQ ID NO: 2944 ttgtagaaatgaaagtaaa 13882 13901 SEQ ID NO: 4282 agcttgtttgccagtgggc 8380 8399 1 5 SEQ ID NO: 2945 ccaaaatttctctcggctt 14011 14030 SEQ ID NO: 4283 taaagccattcaggtctct 3013 3025 1 5 SEQ ID NO: 2946 caaattttctctgctaaaa 14020 14039 SEQ ID NO: 4284 ggacaaggcccagaatctg 9566 9585 1 5 SEQ ID NO: 2947 tctgctggaaacaacaacg 1627 1646 SEQ ID NO: 4285 attcttaacgggattcctt 8641 8660 3 4 SEQ ID NO: 2948 cagaagctaagcaatgtcc 1866 1975 SEQ ID NO: 4286 aattgttgaaagaaaaact 12564 12583 3 4 SEQ ID NO: 2949 taagattaaatatttactt 6435 6454 SEQ ID NO: 4287 agcttgtttgccagtgggc 11409 11426 3 4 SEQ ID NO: 2950 aaagattactttgagaaat 6488 6507 SEQ ID NO: 4288 ttaatctttcataagtcaa 7429 7448 3 4 SEQ ID NO: 2951 aggaaatagttggattacc 26 45 SEQ ID NO: 4289 agcttgtttgccagtgggc 8039 8058 2 4 SEQ ID NO: 2952 attttgatgatgctggccc 253 272 SEQ ID NO: 4290 agcttgtttgccagtgggc 13184 13203 2 4 SEQ ID NO: 2953 gaattatcttttaaacatt 316 335 SEQ ID NO: 4291 attccttccttacaattgg 13415 13434 2 4 SEQ ID NO: 2954 ttaccaccagtttgaagtt 483 502 SEQ ID NO: 4292 agcttgtttgccagtgggc 5889 5908 2 4 SEQ ID NO: 2955 taagattaaatatttactt 555 574 SEQ ID NO: 4293 agcttgtttgccagtgggc 10498 15174 2 4 SEQ ID NO: 2956 aaagattactttgagaaat 1095 1114 SEQ ID NO: 4294 attccttccttacaattgg 13191 13210 2 4 SEQ ID NO: 2957 aggaaatagttggattacc 1210 1220 SEQ ID NO: 4295 tgggctaaacgtttaattg 9237 9256 2 4 SEQ ID NO: 2958 attttgatgatgctggccc 1211 1230 SEQ ID NO: 4296 tattgaaatatgatttttt 9236 9255 2 4 SEQ ID NO: 2959 gaattatcttttaaacatt 1231 1250 SEQ ID NO: 4297 agcttgtttgccagtgggc 5216 5235 2 4 SEQ ID NO: 2960 ttaccaccagtttgaagtt 1628 1647 SEQ ID NO: 4298 tgaaatcattgaaaattta 5918 5994 2 4 SEQ ID NO: 2961 ctgaaaaagttagtgaaag 1948 1988 SEQ ID NO: 4299 caggaggctttaagttgtc 10631 10660 2 4 SEQ ID NO: 2962 tttttcccagacagtgtca 2246 2205 SEQ ID NO: 4300 agcttgtttgccagtgggc 9730 9749 2 4 SEQ ID NO: 2963 ttttcccagacagtgtcca 2247 2268 SEQ ID NO: 4301 taaagccattcaggtctct 9729 9746 2 4 SEQ ID NO: 2964 cattcagaaccagggaaga 3403 3422 SEQ ID NO: 4302 cttcatcgttcatttgagt 10414 10433 2 4 SEQ ID NO: 2965 acaaactgtttatagtccc 3628 3647 SEQ ID NO: 4303 cttatgggatttcctaagg 10902 10921 2 4 SEQ ID NO: 2966 ggatttcaatacatttcac 3844 3663 SEQ ID NO: 4304 cttcatcgttcatttgagt 11815 11834 2 4 SEQ ID NO: 2967 ttgtagaaatgaaagtaaa 4407 4426 SEQ ID NO: 4305 ttccatcacaaatcctttg 7377 7395 2 4 SEQ ID NO: 2968 ccaaaatttctctcggctt 4412 4431 SEQ ID NO: 4306 tctcaaagagttacaacag 7162 7181 2 4 SEQ ID NO: 2969 caaattttctctgctaaaa 5083 5102 SEQ ID NO: 4307 tattgaaatatgatttttt 11384 11403 2 4 SEQ ID NO: 2970 tctgctggaaacaacaacg 5325 5344 SEQ ID NO: 4308 aattgttgaaagaaaaact 7382 7401 2 4 SEQ ID NO: 2971 cagaagctaagcaatgtcc 6074 6093 SEQ ID NO: 4309 ggacaaggcccagaatctg 13876 13896 2 4 SEQ ID NO: 2972 taagattaaatatttactt 6088 6107 SEQ ID NO: 4310 aaagaaacctatggcctta 10087 10704 2 4 SEQ ID NO: 2973 aaagattactttgagaaat 6421 6440 SEQ ID NO: 4311 attcttaacgggattcctt 11456 11505 2 4 SEQ ID NO: 2974 aggaaatagttggattacc 7059 7878 SEQ ID NO: 4312 taaagccattcaggtctct 9381 9400 2 4 SEQ ID NO: 2975 attttgatgatgctggccc 7227 7246 SEQ ID NO: 4313 gacatgtttgaagggtgga 13446 13465 2 4 SEQ ID NO: 2976 gaattatcttttaaacatt 7929 7948 SEQ ID NO: 4314 tgggctaaacgtttaattg 12312 1331 2 4 SEQ ID NO: 2977 ttaccaccagtttgaagtt 8787 8805 SEQ ID NO: 4315 tattgaaatatgatttttt 12533 12552 2 4 SEQ ID NO: 2978 taagattaaatatttactt 10167 10186 SEQ ID NO: 4316 agcttgtttgccagtgggc 13191 13210 2 4 SEQ ID NO: 2979 aaagattactttgagaaat 12898 12917 SEQ ID NO: 4317 tgaaatcattgaaaattta 13640 136659 2 4 SEQ ID NO: 2980 aggaaatagttggattacc 13680 12699 SEQ ID NO: 4318 caggaggctttaagttgtc 13613 13832 2 4 SEQ ID NO: 2981 attttgatgatgctggccc 19 38 SEQ ID NO: 4319 agcttgtttgccagtgggc 84 103 1 4 SEQ ID NO: 2982 aggtttaaatggataattt 20 39 SEQ ID NO: 4320 taaagccattcaggtctct 83 102 1 4 SEQ ID NO: 2983 cagaagctaagcaatgtcc 22 41 SEQ ID NO: 4321 aattgttgaaagaaaaact 11667 11576 1 4 SEQ ID NO: 2984 taagattaaatatttactt 33 52 SEQ ID NO: 4322 taaagccattcaggtctct 2188 2167 1 4 SEQ ID NO: 2985 aaagattactttgagaaat 90 109 SEQ ID NO: 4323 ttaatctttcataagtcaa 376 395 1 4 SEQ ID NO: 2986 aggaaatagttggattacc 151 170 SEQ ID NO: 4324 ggacaaggcccagaatctg 4252 4271 1 4 SEQ ID NO: 2987 attttgatgatgctggccc 177 196 SEQ ID NO: 4325 attcttaacgggattcctt 11186 11202 1 4 SEQ ID NO: 2988 gaattatcttttaaacatt 227 246 SEQ ID NO: 4326 aattgttgaaagaaaaact 388 407 1 4 SEQ ID NO: 2989 ttaccaccagtttgaagtt 291 310 SEQ ID NO: 4327 agcttgtttgccagtgggc 3391 3410 1 4 SEQ ID NO: 2990 ttgcagtgtatcggacaga 269 316 SEQ ID NO: 4328 ttaatctttcataagtcaa 3863 2702 1 4 SEQ ID NO: 2991 cattcagaaccagggaaga 354 373 SEQ ID NO: 4329 agcttgtttgccagtgggc 5736 4755 1 4 SEQ ID NO: 2992 acaaactgtttatagtccc 358 377 SEQ ID NO: 4330 tgaaatcattgaaaattta 9171 9190 1 4 SEQ ID NO: 2993 ggatttcaatacatttcac 375 397 SEQ ID NO: 4331 caggaggctttaagttgtc 3269 3288 1 4 SEQ ID NO: 2994 ttgtagaaatgaaagtaaa 402 421 SEQ ID NO: 4332 agcttgtttgccagtgggc 5802 5821 1 4 SEQ ID NO: 2995 ctgaacagtgagctgcagt 472 491 SEQ ID NO: 4333 taaagccattcaggtctct 6348 6367 1 4 SEQ ID NO: 2996 aatccaatctcctctttgc 600 519 SEQ ID NO: 4334 cttcatcgttcatttgagt 12724 12743 1 4 SEQ ID NO: 2997 atttgattttcaagcaaag 543 582 SEQ ID NO: 4335 cttatgggatttcctaagg 2227 2246 1 4 SEQ ID NO: 2998 ttttgattttcaagcaaat 583 602 SEQ ID NO: 4336 cttcatcgttcatttgagt 2266 2304 1 4 SEQ ID NO: 2999 tgatttcaagcaaatgcag 509 628 SEQ ID NO: 4337 ttccatcacaaatcctttg 13996 14015 1 4 SEQ ID NO: 3000 atgctgtttgggaaattgg 630 649 SEQ ID NO: 4338 tctcaaagagttacaacag 14050 14099 1 4 SEQ ID NO: 3001 tgctgttttttggaaatgc 638 857 SEQ ID NO: 4339 tattgaaatatgatttttt 11050 11049 1 4 SEQ ID NO: 3002 aaaaaaatacactggacgt 652 671 SEQ ID NO: 4340 aattgttgaaagaaaaact 11364 11383 1 4 SEQ ID NO: 3003 actggagcttagtaagggc 678 697 SEQ ID NO: 4341 ggacaaggcccagaatctg 6825 6844 1 4 SEQ ID NO: 3004 cttctggaaagagggtcat 701 720 SEQ ID NO: 4342 aaagaaacctatggcctta 3013 3032 1 4 SEQ ID NO: 3005 ggaaaagggtcatggaaat 708 727 SEQ ID NO: 4343 attcttaacgggattcctt 3466 3507 1 4 SEQ ID NO: 3006 gggcctgccccagatttct 709 728 SEQ ID NO: 4344 taaagccattcaggtctct 13834 13853 1 4 SEQ ID NO: 3007 ttctcagataggggaacac 714 733 SEQ ID NO: 4345 gacatgtttgaagggtgga 1895 1914 1 4 SEQ ID NO: 3008 gatgaaagaattaagggga 737 758 SEQ ID NO: 4346 tgggctaaacgtttaattg 2935 2987 1 4 SEQ ID NO: 3009 cttggactgtcaaataagt 752 771 SEQ ID NO: 4349 tattgaaatatgatttttt 5218 5327 1 4 SEQ ID NO: 3010 ggaaaagggtcatggaaat 783 772 SEQ ID NO: 4350 agcttgtttgccagtgggc 5217 5226 1 4 SEQ ID NO: 3011 gggcctgccccagatttct 784 803 SEQ ID NO: 4351 tgaaatcattgaaaattta 8152 8171 1 4 SEQ ID NO: 3012 cttggactgtcaaataagt 794 813 SEQ ID NO: 4352 tgaaatcattgaaaattta 10345 10364 1 4 SEQ ID NO: 3013 ggatttcaatacatttcac 819 838 SEQ ID NO: 4353 aaagaaacctatggcctta 12453 12472 1 4 SEQ ID NO: 3014 ttgtagaaatgaaagtaaa 820 839 SEQ ID NO: 4354 attcttaacgggattcctt 12452 12471 1 4 SEQ ID NO: 3015 ccaaaatttctctcggctt 892 911 SEQ ID NO: 4355 taaagccattcaggtctct 3813 5832 1 4 SEQ ID NO: 3016 caaattttctctgctaaaa 893 912 SEQ ID NO: 4356 gacatgtttgaagggtgga 3812 3831 1 4 SEQ ID NO: 3017 tctgctggaaacaacaacg 916 935 SEQ ID NO: 4357 tgggctaaacgtttaattg 9481 9480 1 4 SEQ ID NO: 3018 cagaagctaagcaatgtcc 924 943 SEQ ID NO: 4358 tattgaaatatgatttttt 13655 13675 1 4 SEQ ID NO: 3019 taagattaaatatttactt 997 1016 SEQ ID NO: 4359 agcttgtttgccagtgggc 1669 1688 1 4 SEQ ID NO: 3020 aaagattactttgagaaat 998 1017 SEQ ID NO: 4360 tgaaatcattgaaaattta 1668 1687 1 4 SEQ ID NO: 3021 aggaaatagttggattacc 1002 1021 SEQ ID NO: 4361 caggaggctttaagttgtc 9675 9694 1 4 SEQ ID NO: 3022 attttgatgatgctggccc 1031 1050 SEQ ID NO: 4362 agcttgtttgccagtgggc 2077 2096 1 4 SEQ ID NO: 3023 gaattatcttttaaacatt 1090 1109 SEQ ID NO: 4363 taaagccattcaggtctct 5495 5514 1 4 SEQ ID NO: 3024 ttaccaccagtttgaagtt 1094 1113 SEQ ID NO: 4364 aattgttgaaagaaaaact 13192 13211 1 4 SEQ ID NO: 3025 taagattaaatatttactt 1110 1129 SEQ ID NO: 4365 taaagccattcaggtctct 14014 14033 1 4 SEQ ID NO: 3026 aaagattactttgagaaat 1112 1131 SEQ ID NO: 4366 ttaatctttcataagtcaa 4572 4591 1 4 SEQ ID NO: 3027 aggaaatagttggattacc 1117 1136 SEQ ID NO: 4367 ggacaaggcccagaatctg 2578 2597 1 4 SEQ ID NO: 3028 attttgatgatgctggccc 1132 1151 SEQ ID NO: 4368 attcttaacgggattcctt 12208 12228 1 4 SEQ ID NO: 3029 gaattatcttttaaacatt 1162 1181 SEQ ID NO: 4369 aattgttgaaagaaaaact 12273 12292 1 4 SEQ ID NO: 3030 ttaccaccagtttgaagtt 1174 1193 SEQ ID NO: 4370 agcttgtttgccagtgggc 10337 10356 1 4 SEQ ID NO: 3031 ctgaaaaagttagtgaaag 1188 1207 SEQ ID NO: 4371 ttaatctttcataagtcaa 12580 12599 1 4 SEQ ID NO: 3032 tttttcccagacagtgtca 1204 1223 SEQ ID NO: 4372 agcttgtttgccagtgggc 8866 8885 1 4 SEQ ID NO: 3033 ttttcccagacagtgtcca 1210 1229 SEQ ID NO: 4373 tgaaatcattgaaaattta 2169 2166 1 4 SEQ ID NO: 3034 cattcagaaccagggaaga 1211 1230 SEQ ID NO: 4374 caggaggctttaagttgtc 6168 2167 1 4 SEQ ID NO: 3035 acaaactgtttatagtccc 1218 1237 SEQ ID NO: 4375 agcttgtttgccagtgggc 13963 13982 1 4 SEQ ID NO: 3036 ggatttcaatacatttcac 1219 1236 SEQ ID NO: 4376 taaagccattcaggtctct 11248 11267 1 4 SEQ ID NO: 3037 ttgtagaaatgaaagtaaa 1248 1267 SEQ ID NO: 4377 cttcatcgttcatttgagt 8148 8167 1 4 SEQ ID NO: 3038 ccaaaatttctctcggctt 1332 1351 SEQ ID NO: 4378 cttatgggatttcctaagg 10824 10643 1 4 SEQ ID NO: 3039 caaattttctctgctaaaa 1349 1368 SEQ ID NO: 4379 cttcatcgttcatttgagt 3439 3458 1 4 SEQ ID NO: 3040 tctgctggaaacaacaacg 1440 1459 SEQ ID NO: 4380 ttccatcacaaatcctttg 5596 5505 1 4 SEQ ID NO: 3041 cagaagctaagcaatgtcc 1480 1499 SEQ ID NO: 4381 tctcaaagagttacaacag 12355 12374 1 4 SEQ ID NO: 3042 taagattaaatatttactt 1516 1535 SEQ ID NO: 4382 tattgaaatatgatttttt 9938 9957 1 4 SEQ ID NO: 3043 aaagattactttgagaaat 1546 1565 SEQ ID NO: 4383 aattgttgaaagaaaaact 10917 10936 1 4 SEQ ID NO: 3044 aggaaatagttggattacc 1548 1567 SEQ ID NO: 4384 ggacaaggcccagaatctg 8007 6026 1 4 SEQ ID NO: 3045 attttgatgatgctggccc 1600 1579 SEQ ID NO: 4385 aaagaaacctatggcctta 8118 8137 1 4 SEQ ID NO: 3046 gaattatcttttaaacatt 1610 1028 SEQ ID NO: 4386 attcttaacgggattcctt 9024 9043 1 4 SEQ ID NO: 3047 ttaccaccagtttgaagtt 1610 1637 SEQ ID NO: 4387 taaagccattcaggtctct 13727 13748 1 4 SEQ ID NO: 3048 taagattaaatatttactt 1629 1648 SEQ ID NO: 4388 gacatgtttgaagggtgga 1933 1952 1 4 SEQ ID NO: 3049 aaagattactttgagaaat 1703 1722 SEQ ID NO: 4389 tgggctaaacgtttaattg 2485 2504 1 4 SEQ ID NO: 3050 aggaaatagttggattacc 1738 1757 SEQ ID NO: 4390 tattgaaatatgatttttt 5519 5538 1 4 SEQ ID NO: 3051 attttgatgatgctggccc 1744 1783 SEQ ID NO: 4391 agcttgtttgccagtgggc 4439 4458 1 4 SEQ ID NO: 3052 aggtttaaatggataattt 1759 1778 SEQ ID NO: 4392 aaagaaacctatggcctta 8893 6912 1 4 SEQ ID NO: 3053 cagaagctaagcaatgtcc 1781 1800 SEQ ID NO: 4393 attcttaacgggattcctt 9952 9971 1 4 SEQ ID NO: 3054 taagattaaatatttactt 1798 7815 SEQ ID NO: 4394 taaagccattcaggtctct 12045 12084 1 4 SEQ ID NO: 3055 aaagattactttgagaaat 1890 1909 SEQ ID NO: 4395 gacatgtttgaagggtgga 10737 10758 1 4 SEQ ID NO: 3056 aggaaatagttggattacc 1910 1929 SEQ ID NO: 4396 tgggctaaacgtttaattg 14000 14019 1 4 SEQ ID NO: 3057 attttgatgatgctggccc 1913 1932 SEQ ID NO: 4397 tattgaaatatgatttttt 13378 13397 1 4 SEQ ID NO: 3058 gaattatcttttaaacatt 1924 1943 SEQ ID NO: 4398 agcttgtttgccagtgggc 7273 7292 1 4 SEQ ID NO: 3059 ttaccaccagtttgaagtt 1928 1948 SEQ ID NO: 4399 ggacaaggcccagaatctg 13825 13844 1 4 SEQ ID NO: 3060 ttgcagtgtatcggacaga 1930 1949 SEQ ID NO: 4400 aaagaaacctatggcctta 13824 13843 1 4 SEQ ID NO: 3061 cattcagaaccagggaaga 1935 1954 SEQ ID NO: 4401 attcttaacgggattcctt 8325 8344 1 4 SEQ ID NO: 3062 acaaactgtttatagtccc 1938 1957 SEQ ID NO: 4402 taaagccattcaggtctct 10185 10204 1 4 SEQ ID NO: 3063 ttaccaccagtttgaagtt 1939 1956 SEQ ID NO: 4403 aattgttgaaagaaaaact 10184 10203 1 4 SEQ ID NO: 3064 taagattaaatatttactt 1943 1982 SEQ ID NO: 4404 ggacaaggcccagaatctg 4973 4982 1 4 SEQ ID NO: 3065 aaagattactttgagaaat 1944 1953 SEQ ID NO: 4405 aaagaaacctatggcctta 4972 4991 1 4 SEQ ID NO: 3066 aggaaatagttggattacc 1950 1969 SEQ ID NO: 4406 attcttaacgggattcctt 10630 10649 1 4 SEQ ID NO: 3067 attttgatgatgctggccc 1990 2009 SEQ ID NO: 4407 taaagccattcaggtctct 13945 13964 1 4 SEQ ID NO: 3068 aggtttaaatggataattt 2007 2026 SEQ ID NO: 4408 gacatgtttgaagggtgga 9501 9520 1 4 SEQ ID NO: 3069 cagaagctaagcaatgtcc 2052 2071 SEQ ID NO: 4409 tgggctaaacgtttaattg 8441 9460 1 4 SEQ ID NO: 3070 taagattaaatatttactt 2065 2084 SEQ ID NO: 4410 tattgaaatatgatttttt 7820 7839 1 4 SEQ ID NO: 3071 aaagattactttgagaaat 2096 2087 SEQ ID NO: 4411 agcttgtttgccagtgggc 7822 7841 1 4 SEQ ID NO: 3072 aggaaatagttggattacc 2091 2110 SEQ ID NO: 4412 tgaaatcattgaaaattta 11621 11840 1 4 SEQ ID NO: 3073 attttgatgatgctggccc 2092 2111 SEQ ID NO: 4413 caggaggctttaagttgtc 14019 14038 1 4 SEQ ID NO: 3074 gaattatcttttaaacatt 117 2136 SEQ ID NO: 4414 agcttgtttgccagtgggc 3614 3633 1 4 SEQ ID NO: 3075 ttaccaccagtttgaagtt 2121 2140 SEQ ID NO: 4415 taaagccattcaggtctct 6694 6713 1 4 SEQ ID NO: 3076 ttgcagtgtatcggacaga 2122 2141 SEQ ID NO: 4416 aattgttgaaagaaaaact 9490 9509 1 4 SEQ ID NO: 3077 cattcagaaccagggaaga 2183 2202 SEQ ID NO: 4417 taaagccattcaggtctct 11709 11728 1 4 SEQ ID NO: 3078 acaaactgtttatagtccc 2206 2225 SEQ ID NO: 4418 ttaatctttcataagtcaa 11066 11087 1 4 SEQ ID NO: 3079 ggatttcaatacatttcac 2253 2272 SEQ ID NO: 4419 ggacaaggcccagaatctg 6142 6161 1 4 SEQ ID NO: 3080 ttgtagaaatgaaagtaaa 2257 2276 SEQ ID NO: 4420 attcttaacgggattcctt 9857 9675 1 4 SEQ ID NO: 3081 ctgaacagtgagctgcagt 2258 2277 SEQ ID NO: 4421 aattgttgaaagaaaaact 4359 4378 1 4 SEQ ID NO: 3082 aatccaatctcctctttgc 2298 2317 SEQ ID NO: 4422 agcttgtttgccagtgggc 3333 3352 1 4 SEQ ID NO: 3083 atttgattttcaagcaaag 2299 2318 SEQ ID NO: 4423 ttaatctttcataagtcaa 8831 8850 1 4 SEQ ID NO: 3084 ttttgattttcaagcaaat 2351 2370 SEQ ID NO: 4424 agcttgtttgccagtgggc 6796 6815 1 4 SEQ ID NO: 3085 tgatttcaagcaaatgcag 2395 2414 SEQ ID NO: 4425 tgaaatcattgaaaattta 6287 5306 1 4 SEQ ID NO: 3086 atgctgtttgggaaattgg 2405 2424 SEQ ID NO: 4426 caggaggctttaagttgtc 7614 7633 1 4 SEQ ID NO: 3087 tgctgttttttggaaatgc 2518 2537 SEQ ID NO: 4427 agcttgtttgccagtgggc 7983 8002 1 4 SEQ ID NO: 3088 aaaaaaatacactggacgt 2540 2559 SEQ ID NO: 4428 taaagccattcaggtctct 9083 9102 1 4 SEQ ID NO: 3089 actggagcttagtaagggc 2593 2612 SEQ ID NO: 4429 cttcatcgttcatttgagt 10382 10401 1 4 SEQ ID NO: 3090 cttctggaaagagggtcat 2596 2615 SEQ ID NO: 4430 cttatgggatttcctaagg 3615 3634 1 4 SEQ ID NO: 3091 ggaaaagggtcatggaaat 2603 2622 SEQ ID NO: 4431 cttcatcgttcatttgagt 9445 9464 1 4 SEQ ID NO: 3092 gggcctgccccagatttct 2609 2828 SEQ ID NO: 4432 ttccatcacaaatcctttg 8607 6628 1 4 SEQ ID NO: 3093 ttctcagataggggaacac 2610 2629 SEQ ID NO: 4433 tctcaaagagttacaacag 13116 13135 1 4 SEQ ID NO: 3094 gatgaaagaattaagggga 2624 2643 SEQ ID NO: 4434 tattgaaatatgatttttt 9842 9661 1 4 SEQ ID NO: 3095 cttggactgtcaaataagt 2625 2644 SEQ ID NO: 4435 aattgttgaaagaaaaact 9641 9660 1 4 SEQ ID NO: 3096 ggaaaagggtcatggaaat 2526 2645 SEQ ID NO: 4436 ggacaaggcccagaatctg 9640 9859 1 4 SEQ ID NO: 3097 gggcctgccccagatttct 2635 2054 SEQ ID NO: 4437 aaagaaacctatggcctta 9344 9363 1 4 SEQ ID NO: 3098 cttggactgtcaaataagt 2638 2655 SEQ ID NO: 4438 attcttaacgggattcctt 9343 9362 1 4 SEQ ID NO: 3099 ggatttcaatacatttcac 2652 2671 SEQ ID NO: 4439 taaagccattcaggtctct 5699 6618 1 4 SEQ ID NO: 3100 ttgtagaaatgaaagtaaa 2653 2872 SEQ ID NO: 4440 gacatgtttgaagggtgga 6598 6617 1 4 SEQ ID NO: 3101 ccaaaatttctctcggctt 2658 2877 SEQ ID NO: 4441 tgggctaaacgtttaattg 14004 14023 1 4 SEQ ID NO: 3102 caaattttctctgctaaaa 2705 2722 SEQ ID NO: 4442 tattgaaatatgatttttt 7600 7619 1 4 SEQ ID NO: 3103 tctgctggaaacaacaacg 2728 2747 SEQ ID NO: 4443 agcttgtttgccagtgggc 9228 9247 1 4 SEQ ID NO: 3104 cagaagctaagcaatgtcc 2758 2777 SEQ ID NO: 4444 tgaaatcattgaaaattta 8538 8557 1 4 SEQ ID NO: 3105 taagattaaatatttactt 2786 2785 SEQ ID NO: 4445 tgaaatcattgaaaattta 9521 9540 1 4 SEQ ID NO: 3106 aaagattactttgagaaat 2819 2838 SEQ ID NO: 4446 aaagaaacctatggcctta 2877 2896 1 4 SEQ ID NO: 3107 aggaaatagttggattacc 2833 2852 SEQ ID NO: 4447 attcttaacgggattcctt 5235 5254 1 4 SEQ ID NO: 3108 attttgatgatgctggccc 2840 2859 SEQ ID NO: 4448 taaagccattcaggtctct 6140 6169 1 4 SEQ ID NO: 3109 gaattatcttttaaacatt 2866 2985 SEQ ID NO: 4449 gacatgtttgaagggtgga 12120 13139 1 4 SEQ ID NO: 3110 ttaccaccagtttgaagtt 2872 2981 SEQ ID NO: 4450 tgggctaaacgtttaattg 5463 5482 1 4 SEQ ID NO: 3111 taagattaaatatttactt 3114 3133 SEQ ID NO: 4451 tattgaaatatgatttttt 12726 12746 1 4 SEQ ID NO: 3112 aaagattactttgagaaat 3206 3227 SEQ ID NO: 4452 agcttgtttgccagtgggc 8393 88412 1 4 SEQ ID NO: 3113 aggaaatagttggattacc 3252 3271 SEQ ID NO: 4453 tgaaatcattgaaaattta 3817 3836 1 4 SEQ ID NO: 3114 attttgatgatgctggccc 3297 3316 SEQ ID NO: 4454 caggaggctttaagttgtc 8357 6376 1 4 SEQ ID NO: 3115 gaattatcttttaaacatt 3313 3332 SEQ ID NO: 4455 agcttgtttgccagtgggc 8938 8957 1 4 SEQ ID NO: 3116 ttaccaccagtttgaagtt 3315 3334 SEQ ID NO: 4456 taaagccattcaggtctct 13207 13226 1 4 SEQ ID NO: 3117 ctgaaaaagttagtgaaag 3337 3356 SEQ ID NO: 4457 aattgttgaaagaaaaact 4211 4230 1 4 SEQ ID NO: 3118 tttttcccagacagtgtca 3345 3364 SEQ ID NO: 4458 taaagccattcaggtctct 7593 7612 1 4 SEQ ID NO: 3119 ttttcccagacagtgtcca 3392 3411 SEQ ID NO: 4459 ttaatctttcataagtcaa 12439 12458 1 4 SEQ ID NO: 3120 cattcagaaccagggaaga 3403 3422 SEQ ID NO: 4460 ggacaaggcccagaatctg 8104 8123 1 4 SEQ ID NO: 3121 acaaactgtttatagtccc 3422 3441 SEQ ID NO: 4461 attcttaacgggattcctt 10924 10943 1 4 SEQ ID NO: 3122 ggatttcaatacatttcac 3486 3505 SEQ ID NO: 4462 aattgttgaaagaaaaact 13800 13899 1 4 SEQ ID NO: 3123 ttgtagaaatgaaagtaaa 3501 3520 SEQ ID NO: 4463 agcttgtttgccagtgggc 5408 5427 1 4 SEQ ID NO: 3124 ccaaaatttctctcggctt 3502 3521 SEQ ID NO: 4464 ggacaaggcccagaatctg 5272 5291 1 4 SEQ ID NO: 3125 caaattttctctgctaaaa 3503 3522 SEQ ID NO: 4465 aaagaaacctatggcctta 5271 5290 1 4 SEQ ID NO: 3126 tctgctggaaacaacaacg 3554 3573 SEQ ID NO: 4466 attcttaacgggattcctt 4567 4536 1 4 SEQ ID NO: 3127 cagaagctaagcaatgtcc 3577 3526 SEQ ID NO: 4467 taaagccattcaggtctct 7809 7626 1 4 SEQ ID NO: 3128 taagattaaatatttactt 3581 3600 SEQ ID NO: 4468 gacatgtttgaagggtgga 5897 6916 1 4 SEQ ID NO: 3129 aaagattactttgagaaat 3600 3619 SEQ ID NO: 4469 tgggctaaacgtttaattg 8877 8895 1 4 SEQ ID NO: 3130 aggaaatagttggattacc 3603 3622 SEQ ID NO: 4470 tattgaaatatgatttttt 9361 8380 1 4 SEQ ID NO: 3131 attttgatgatgctggccc 3811 3630 SEQ ID NO: 4471 agcttgtttgccagtgggc 8071 8090 1 4 SEQ ID NO: 3132 gaattatcttttaaacatt 3625 3644 SEQ ID NO: 4472 tgaaatcattgaaaattta 7871 7880 1 4 SEQ ID NO: 3133 ttaccaccagtttgaagtt 6289 3646 SEQ ID NO: 4473 tgaaatcattgaaaattta 5287 5308 1 4 SEQ ID NO: 3134 taagattaaatatttactt 3632 3651 SEQ ID NO: 4474 aaagaaacctatggcctta 8278 8297 1 4 SEQ ID NO: 3135 aaagattactttgagaaat 3844 3003 SEQ ID NO: 4475 attcttaacgggattcctt 11236 11255 1 4 SEQ ID NO: 3136 aggaaatagttggattacc 3658 3677 SEQ ID NO: 4476 taaagccattcaggtctct 8591 5610 1 4 SEQ ID NO: 3137 attttgatgatgctggccc 3776 3695 SEQ ID NO: 4477 gacatgtttgaagggtgga 8366 8385 1 4 SEQ ID NO: 3138 aggtttaaatggataattt 3878 3696 SEQ ID NO: 4478 tgggctaaacgtttaattg 8355 8384 1 4 SEQ ID NO: 3139 ggatttcaatacatttcac 3678 3697 SEQ ID NO: 4479 tattgaaatatgatttttt 8406 8425 1 4 SEQ ID NO: 3140 ttgtagaaatgaaagtaaa 3760 3779 SEQ ID NO: 4480 agcttgtttgccagtgggc 4090 4109 1 4 SEQ ID NO: 3141 ctgaacagtgagctgcagt 3803 3822 SEQ ID NO: 4481 tgaaatcattgaaaattta 13186 13205 1 4 SEQ ID NO: 3142 aatccaatctcctctttgc 3899 3918 SEQ ID NO: 4482 caggaggctttaagttgtc 8142 8161 1 4 SEQ ID NO: 3143 atttgattttcaagcaaag 3915 3934 SEQ ID NO: 4483 agcttgtttgccagtgggc 8003 8922 1 4 SEQ ID NO: 3144 ttttgattttcaagcaaat 3994 4013 SEQ ID NO: 4484 taaagccattcaggtctct 9541 9550 1 4 SEQ ID NO: 3145 acaaactgtttatagtccc 4000 4019 SEQ ID NO: 4485 aattgttgaaagaaaaact 13604 13713 1 4 SEQ ID NO: 3146 ggatttcaatacatttcac 4015 4034 SEQ ID NO: 4486 taaagccattcaggtctct 6359 6378 1 4 SEQ ID NO: 3147 ttgtagaaatgaaagtaaa 4036 4055 SEQ ID NO: 4487 ttaatctttcataagtcaa 7504 7613 1 4 SEQ ID NO: 3148 ttaccaccagtttgaagtt 4045 4064 SEQ ID NO: 4488 ggacaaggcccagaatctg 10033 10052 1 4 SEQ ID NO: 3149 ttgcagtgtatcggacaga 4062 4111 SEQ ID NO: 4489 attcttaacgggattcctt 9735 9754 1 4 SEQ ID NO: 3150 cattcagaaccagggaaga 4104 4123 SEQ ID NO: 4490 aattgttgaaagaaaaact 8558 8576 1 4 SEQ ID NO: 3151 acaaactgtttatagtccc 4112 4131 SEQ ID NO: 4491 agcttgtttgccagtgggc 8215 8234 1 4 SEQ ID NO: 3152 ggatttcaatacatttcac 4126 4145 SEQ ID NO: 4492 ttaatctttcataagtcaa 6088 6105 1 4 SEQ ID NO: 3153 ttgtagaaatgaaagtaaa 4133 4152 SEQ ID NO: 4493 agcttgtttgccagtgggc 8109 8128 1 4 SEQ ID NO: 3154 ccaaaatttctctcggctt 4141 4160 SEQ ID NO: 4494 tgaaatcattgaaaattta 12009 12028 1 4 SEQ ID NO: 3155 caaattttctctgctaaaa 4284 4303 SEQ ID NO: 4495 caggaggctttaagttgtc 9024 9043 1 4 SEQ ID NO: 3156 tctgctggaaacaacaacg 4317 4336 SEQ ID NO: 4496 agcttgtttgccagtgggc 9503 9822 1 4 SEQ ID NO: 3157 cagaagctaagcaatgtcc 4338 4357 SEQ ID NO: 4497 taaagccattcaggtctct 11035 11054 1 4 SEQ ID NO: 3158 taagattaaatatttactt 4376 4397 SEQ ID NO: 4498 cttcatcgttcatttgagt 8585 6584 1 4 SEQ ID NO: 3159 aaagattactttgagaaat 4380 4399 SEQ ID NO: 4499 cttatgggatttcctaagg 12133 12152 1 4 SEQ ID NO: 3160 aggaaatagttggattacc 4407 4426 SEQ ID NO: 4500 cttcatcgttcatttgagt 7300 7327 1 4 SEQ ID NO: 3161 attttgatgatgctggccc 4499 4518 SEQ ID NO: 4501 ttccatcacaaatcctttg 8334 8353 1 4 SEQ ID NO: 3162 gaattatcttttaaacatt 4644 4663 SEQ ID NO: 4502 tctcaaagagttacaacag 8954 7009 1 4 SEQ ID NO: 3163 ttaccaccagtttgaagtt 4645 4664 SEQ ID NO: 4503 tattgaaatatgatttttt 6963 7002 1 4 SEQ ID NO: 3164 taagattaaatatttactt 4646 4665 SEQ ID NO: 4504 aattgttgaaagaaaaact 6982 7001 1 4 SEQ ID NO: 3165 aaagattactttgagaaat 4650 4889 SEQ ID NO: 4505 ggacaaggcccagaatctg 12073 12092 1 4 SEQ ID NO: 3166 aggaaatagttggattacc 4713 4732 SEQ ID NO: 4506 aaagaaacctatggcctta 10210 10229 1 4 SEQ ID NO: 3167 attttgatgatgctggccc 4745 4764 SEQ ID NO: 4507 attcttaacgggattcctt 8184 8203 1 4 SEQ ID NO: 3168 gaattatcttttaaacatt 4818 4837 SEQ ID NO: 4508 taaagccattcaggtctct 7930 7949 1 4 SEQ ID NO: 3169 ttaccaccagtttgaagtt 4820 4839 SEQ ID NO: 4509 gacatgtttgaagggtgga 8740 8759 1 4 SEQ ID NO: 3170 ctgaaaaagttagtgaaag 4873 4892 SEQ ID NO: 4510 tgggctaaacgtttaattg 6843 6862 1 4 SEQ ID NO: 3171 tttttcccagacagtgtca 4909 4926 SEQ ID NO: 4511 tattgaaatatgatttttt 9350 9369 1 4 SEQ ID NO: 3172 ttttcccagacagtgtcca 4913 4932 SEQ ID NO: 4512 agcttgtttgccagtgggc 8511 6630 1 4 SEQ ID NO: 3173 cattcagaaccagggaaga 4976 4995 SEQ ID NO: 4513 aaagaaacctatggcctta 12272 12291 1 4 SEQ ID NO: 3174 acaaactgtttatagtccc 4984 5003 SEQ ID NO: 4514 attcttaacgggattcctt 10809 10828 1 4 SEQ ID NO: 3175 ggatttcaatacatttcac 4988 5007 SEQ ID NO: 4515 taaagccattcaggtctct 10042 10061 1 4 SEQ ID NO: 3176 ttgtagaaatgaaagtaaa 4989 5008 SEQ ID NO: 4516 gacatgtttgaagggtgga 10041 10080 1 4 SEQ ID NO: 3177 ccaaaatttctctcggctt 4990 5009 SEQ ID NO: 4517 tgggctaaacgtttaattg 4733 4752 1 4 SEQ ID NO: 3178 caaattttctctgctaaaa 8091 5113 SEQ ID NO: 4518 tattgaaatatgatttttt 12267 12266 1 4 SEQ ID NO: 3179 tctgctggaaacaacaacg 5100 5119 SEQ ID NO: 4519 agcttgtttgccagtgggc 12514 12533 1 4 SEQ ID NO: 3180 cagaagctaagcaatgtcc 5114 5133 SEQ ID NO: 4520 ggacaaggcccagaatctg 7734 7753 1 4 SEQ ID NO: 3181 taagattaaatatttactt 5151 5170 SEQ ID NO: 4521 aaagaaacctatggcctta 13579 13598 1 4 SEQ ID NO: 3182 aaagattactttgagaaat 5178 5107 SEQ ID NO: 4522 attcttaacgggattcctt 8649 8668 1 4 SEQ ID NO: 3183 aggaaatagttggattacc 5207 5220 SEQ ID NO: 4523 taaagccattcaggtctct 9727 9746 1 4 SEQ ID NO: 3184 attttgatgatgctggccc 5265 5284 SEQ ID NO: 4524 aattgttgaaagaaaaact 5838 5857 1 4 SEQ ID NO: 3185 gaattatcttttaaacatt 5289 5308 SEQ ID NO: 4525 ggacaaggcccagaatctg 6993 7012 1 4 SEQ ID NO: 3186 ttaccaccagtttgaagtt 5324 5434 SEQ ID NO: 4526 aaagaaacctatggcctta 7255 7274 1 4 SEQ ID NO: 3187 taagattaaatatttactt 5325 5344 SEQ ID NO: 4527 attcttaacgggattcctt 7254 7273 1 4 SEQ ID NO: 3188 aaagattactttgagaaat 5341 6360 SEQ ID NO: 4528 taaagccattcaggtctct 7624 7643 1 4 SEQ ID NO: 3189 aggaaatagttggattacc 5346 6365 SEQ ID NO: 4529 gacatgtttgaagggtgga 12942 12961 1 4 SEQ ID NO: 3190 attttgatgatgctggccc 5407 6428 SEQ ID NO: 4530 tgggctaaacgtttaattg 8380 3399 1 4 SEQ ID NO: 3191 aggtttaaatggataattt 5412 6431 SEQ ID NO: 4531 tattgaaatatgatttttt 8504 5523 1 4 SEQ ID NO: 3192 cagaagctaagcaatgtcc 5445 5464 SEQ ID NO: 4532 agcttgtttgccagtgggc 9593 9712 1 4 SEQ ID NO: 3193 taagattaaatatttactt 5450 5469 SEQ ID NO: 4533 tgaaatcattgaaaattta 4429 4448 1 4 SEQ ID NO: 3194 aaagattactttgagaaat 5465 5485 SEQ ID NO: 4534 caggaggctttaagttgtc 6388 6407 1 4 SEQ ID NO: 3195 aggaaatagttggattacc 5474 5493 SEQ ID NO: 4535 agcttgtttgccagtgggc 6425 6444 1 4 SEQ ID NO: 3196 attttgatgatgctggccc 5494 8513 SEQ ID NO: 4536 taaagccattcaggtctct 11820 11839 1 4 SEQ ID NO: 3197 gaattatcttttaaacatt 5506 8825 SEQ ID NO: 4537 aattgttgaaagaaaaact 7032 7051 1 4 SEQ ID NO: 3198 ttaccaccagtttgaagtt 5507 8826 SEQ ID NO: 4538 taaagccattcaggtctct 10434 10453 1 4 SEQ ID NO: 3199 ttgcagtgtatcggacaga 5512 8831 SEQ ID NO: 4539 ttaatctttcataagtcaa 6131 6150 1 4 SEQ ID NO: 3200 cattcagaaccagggaaga 5554 5803 SEQ ID NO: 4540 ggacaaggcccagaatctg 10765 10784 1 4 SEQ ID NO: 3201 acaaactgtttatagtccc 5857 5676 SEQ ID NO: 4541 attcttaacgggattcctt 12764 12783 1 4 SEQ ID NO: 3202 ggatttcaatacatttcac 5692 5711 SEQ ID NO: 4542 aattgttgaaagaaaaact 9014 9033 1 4 SEQ ID NO: 3203 ttgtagaaatgaaagtaaa 5775 5794 SEQ ID NO: 4543 agcttgtttgccagtgggc 8437 8456 1 4 SEQ ID NO: 3204 ctgaacagtgagctgcagt 5776 5795 SEQ ID NO: 4544 ttaatctttcataagtcaa 13312 13331 1 4 SEQ ID NO: 3205 aatccaatctcctctttgc 5848 5867 SEQ ID NO: 4545 taaagccattcaggtctct 11176 11195 1 4 SEQ ID NO: 3206 atttgattttcaagcaaag 5896 5915 SEQ ID NO: 4546 aattgttgaaagaaaaact 10916 10938 1 4 SEQ ID NO: 3207 ttttgattttcaagcaaat 66043 6062 SEQ ID NO: 4547 taaagccattcaggtctct 8341 8360 1 4 SEQ ID NO: 3208 tgatttcaagcaaatgcag 6053 6072 SEQ ID NO: 4548 ttaatctttcataagtcaa 11221 11240 1 4 SEQ ID NO: 3209 atgctgtttgggaaattgg 8088 6107 SEQ ID NO: 4549 ggacaaggcccagaatctg 9330 8398 1 4 SEQ ID NO: 3210 tgctgttttttggaaatgc 6089 6108 SEQ ID NO: 4550 attcttaacgggattcctt 10656 10687 1 4 SEQ ID NO: 3211 aaaaaaatacactggacgt 8147 6165 SEQ ID NO: 4551 aattgttgaaagaaaaact 7930 7958 1 4 SEQ ID NO: 3212 actggagcttagtaagggc 8201 6220 SEQ ID NO: 4552 agcttgtttgccagtgggc 8876 6897 1 4 SEQ ID NO: 3213 cttctggaaagagggtcat 6241 6260 SEQ ID NO: 4553 ttaatctttcataagtcaa 10209 10228 1 4 SEQ ID NO: 3214 ggaaaagggtcatggaaat 6277 6296 SEQ ID NO: 4554 agcttgtttgccagtgggc 9064 9083 1 4 SEQ ID NO: 3215 gggcctgccccagatttct 6285 6304 SEQ ID NO: 4555 tgaaatcattgaaaattta 13318 13337 1 4 SEQ ID NO: 3216 ttctcagataggggaacac 287 6306 SEQ ID NO: 4556 caggaggctttaagttgtc 8421 8440 1 4 SEQ ID NO: 3217 gatgaaagaattaagggga 6320 6339 SEQ ID NO: 4557 agcttgtttgccagtgggc 10727 10746 1 4 SEQ ID NO: 3218 cttggactgtcaaataagt 6321 6340 SEQ ID NO: 4558 taaagccattcaggtctct 8893 8912 1 4 SEQ ID NO: 3219 ggaaaagggtcatggaaat 6346 6365 SEQ ID NO: 4559 cttcatcgttcatttgagt 8528 8547 1 4 SEQ ID NO: 3220 aggaaatagttggattacc 6352 6371 SEQ ID NO: 4560 cttatgggatttcctaagg 11708 11727 1 4 SEQ ID NO: 3221 attttgatgatgctggccc 6380 6399 SEQ ID NO: 4561 cttcatcgttcatttgagt 10435 10454 1 4 SEQ ID NO: 3222 gaattatcttttaaacatt 6415 6434 SEQ ID NO: 4562 ttccatcacaaatcctttg 7999 8018 1 4 SEQ ID NO: 3223 ttaccaccagtttgaagtt 3420 6439 SEQ ID NO: 4563 tctcaaagagttacaacag 11467 11508 1 4 SEQ ID NO: 3224 ctgaaaaagttagtgaaag 6455 6484 SEQ ID NO: 4564 tattgaaatatgatttttt 10000 10079 1 4 SEQ ID NO: 3225 tttttcccagacagtgtca 6494 6613 SEQ ID NO: 4565 aattgttgaaagaaaaact 11371 11390 1 4 SEQ ID NO: 3226 ttttcccagacagtgtcca 6405 6514 SEQ ID NO: 4566 ggacaaggcccagaatctg 11370 11359 1 4 SEQ ID NO: 3227 cattcagaaccagggaaga 6540 6559 SEQ ID NO: 4567 aaagaaacctatggcctta 7967 8008 1 4 SEQ ID NO: 3228 acaaactgtttatagtccc 6558 6577 SEQ ID NO: 4568 attcttaacgggattcctt 9050 9069 1 4 SEQ ID NO: 3229 ggatttcaatacatttcac 6611 6630 SEQ ID NO: 4569 taaagccattcaggtctct 6811 6830 1 4 SEQ ID NO: 3230 ttgtagaaatgaaagtaaa 6694 5713 SEQ ID NO: 4570 gacatgtttgaagggtgga 6710 8735 1 4 SEQ ID NO: 3231 ccaaaatttctctcggctt 8898 5715 SEQ ID NO: 4571 tgggctaaacgtttaattg 10441 10460 1 4 SEQ ID NO: 3232 caaattttctctgctaaaa 8710 5729 SEQ ID NO: 4572 tattgaaatatgatttttt 10159 10178 1 4 SEQ ID NO: 3233 tctgctggaaacaacaacg 8737 5766 SEQ ID NO: 4573 agcttgtttgccagtgggc 11161 11180 1 4 SEQ ID NO: 3234 cagaagctaagcaatgtcc 8762 6781 SEQ ID NO: 4574 aaagaaacctatggcctta 7281 7300 1 4 SEQ ID NO: 3235 taagattaaatatttactt 8823 6842 SEQ ID NO: 4575 attcttaacgggattcctt 11404 11423 1 4 SEQ ID NO: 3236 aaagattactttgagaaat 8914 6933 SEQ ID NO: 4576 taaagccattcaggtctct 10699 10716 1 4 SEQ ID NO: 3237 aggaaatagttggattacc 8973 6992 SEQ ID NO: 4577 gacatgtttgaagggtgga 10571 10690 1 4 SEQ ID NO: 3238 attttgatgatgctggccc 7059 7078 SEQ ID NO: 4578 tgggctaaacgtttaattg 7104 7123 1 4 SEQ ID NO: 3239 gaattatcttttaaacatt 7100 7119 SEQ ID NO: 4579 tattgaaatatgatttttt 8421 8440 1 4 SEQ ID NO: 3240 ttaccaccagtttgaagtt 7199 7218 SEQ ID NO: 4580 agcttgtttgccagtgggc 7776 7796 1 4 SEQ ID NO: 3241 taagattaaatatttactt 7227 7245 SEQ ID NO: 4581 ggacaaggcccagaatctg 8526 8545 1 4 SEQ ID NO: 3242 aaagattactttgagaaat 7232 7251 SEQ ID NO: 4582 aaagaaacctatggcctta 10301 10320 1 4 SEQ ID NO: 3243 aggaaatagttggattacc 7293 7312 SEQ ID NO: 4583 attcttaacgggattcctt 7320 7339 1 4 SEQ ID NO: 3244 attttgatgatgctggccc 7327 7346 SEQ ID NO: 4584 taaagccattcaggtctct 13209 13228 1 4 SEQ ID NO: 3245 aggtttaaatggataattt 7365 7334 SEQ ID NO: 4585 taaagccattcaggtctct 7254 7273 1 4 SEQ ID NO: 3246 cagaagctaagcaatgtcc 7394 7413 SEQ ID NO: 4586 gacatgtttgaagggtgga 8723 9742 1 4 SEQ ID NO: 3247 taagattaaatatttactt 7484 7433 SEQ ID NO: 4587 tgggctaaacgtttaattg 8076 9095 1 4 SEQ ID NO: 3248 aaagattactttgagaaat 7475 7494 SEQ ID NO: 4588 tattgaaatatgatttttt 8526 8544 1 4 SEQ ID NO: 3249 aggaaatagttggattacc 7492 7511 SEQ ID NO: 4589 agcttgtttgccagtgggc 10645 10064 1 4 SEQ ID NO: 3250 attttgatgatgctggccc 7576 7597 SEQ ID NO: 4590 ggacaaggcccagaatctg 10950 10096 1 4 SEQ ID NO: 3251 gaattatcttttaaacatt 7551 7600 SEQ ID NO: 4591 aaagaaacctatggcctta 10749 10768 1 4 SEQ ID NO: 3252 ttaccaccagtttgaagtt 7815 7634 SEQ ID NO: 4592 attcttaacgggattcctt 9480 9499 1 4 SEQ ID NO: 3253 ttgcagtgtatcggacaga 7739 7758 SEQ ID NO: 4593 taaagccattcaggtctct 7805 7884 1 4 SEQ ID NO: 3254 cattcagaaccagggaaga 7870 7889 SEQ ID NO: 4594 tattgaaatatgatttttt 8922 8941 1 4 SEQ ID NO: 3255 acaaactgtttatagtccc 7873 7892 SEQ ID NO: 4595 agcttgtttgccagtgggc 8006 9027 1 4 SEQ ID NO: 3256 ggatttcaatacatttcac 7874 7993 SEQ ID NO: 4596 ggacaaggcccagaatctg 6007 9026 1 4 SEQ ID NO: 3257 ttgtagaaatgaaagtaaa 7909 7928 SEQ ID NO: 4597 aaagaaacctatggcctta 12580 12599 1 4 SEQ ID NO: 3258 ctgaacagtgagctgcagt 7980 7999 SEQ ID NO: 4598 taaagccattcaggtctct 13222 13241 1 4 SEQ ID NO: 3259 aatccaatctcctctttgc 8080 8069 SEQ ID NO: 4599 taaagccattcaggtctct 8418 9437 1 4 SEQ ID NO: 3260 atttgattttcaagcaaag 8136 8155 SEQ ID NO: 4600 gacatgtttgaagggtgga 12438 12457 1 4 SEQ ID NO: 3261 ttttgattttcaagcaaat 8226 8245 SEQ ID NO: 4601 tgggctaaacgtttaattg 12301 12320 1 4 SEQ ID NO: 3262 tgatttcaagcaaatgcag 8299 6318 SEQ ID NO: 4602 tgggctaaacgtttaattg 12579 12508 1 4 SEQ ID NO: 3263 atgctgtttgggaaattgg 8328 8347 SEQ ID NO: 4603 attcttaacgggattcctt 10026 10045 1 4 SEQ ID NO: 3264 tgctgttttttggaaatgc 8436 8457 SEQ ID NO: 4604 aattgttgaaagaaaaact 10005 10024 1 4 SEQ ID NO: 3265 aaaaaaatacactggacgt 8439 8458 SEQ ID NO: 4605 agcttgtttgccagtgggc 9245 9254 1 4 SEQ ID NO: 3266 ctgaaaaagttagtgaaag 8458 8477 SEQ ID NO: 4606 ttaatctttcataagtcaa 9291 9310 1 4 SEQ ID NO: 3267 tttttcccagacagtgtca 8476 8495 SEQ ID NO: 4607 agcttgtttgccagtgggc 10972 10991 1 4 SEQ ID NO: 3268 ttttcccagacagtgtcca 8551 8570 SEQ ID NO: 4608 tgaaatcattgaaaattta 13501 13520 1 4 SEQ ID NO: 3269 cattcagaaccagggaaga 8592 8611 SEQ ID NO: 4609 caggaggctttaagttgtc 13788 13807 1 4 SEQ ID NO: 3270 acaaactgtttatagtccc 8652 8671 SEQ ID NO: 4610 agcttgtttgccagtgggc 13194 13213 1 4 SEQ ID NO: 3271 ggatttcaatacatttcac 8729 8748 SEQ ID NO: 4611 taaagccattcaggtctct 9231 9250 1 4 SEQ ID NO: 3272 ttgtagaaatgaaagtaaa 8741 8760 SEQ ID NO: 4612 cttcatcgttcatttgagt 4819 4838 1 4 SEQ ID NO: 3273 ccaaaatttctctcggctt 8787 8808 SEQ ID NO: 4613 cttatgggatttcctaagg 11172 11191 1 4 SEQ ID NO: 3274 caaattttctctgctaaaa 8795 8814 SEQ ID NO: 4614 cttcatcgttcatttgagt 12603 12622 1 4 SEQ ID NO: 3275 tctgctggaaacaacaacg 8722 8818 SEQ ID NO: 4615 ttccatcacaaatcctttg 9090 9109 1 4 SEQ ID NO: 3276 cagaagctaagcaatgtcc 8814 8833 SEQ ID NO: 4616 tctcaaagagttacaacag 11010 11009 1 4 SEQ ID NO: 3277 taagattaaatatttactt 8944 8983 SEQ ID NO: 4617 tattgaaatatgatttttt 12817 12836 1 4 SEQ ID NO: 3278 aaagattactttgagaaat 8988 8957 SEQ ID NO: 4618 aattgttgaaagaaaaact 12292 12311 1 4 SEQ ID NO: 3279 aggaaatagttggattacc 8988 9007 SEQ ID NO: 4619 ggacaaggcccagaatctg 11634 11653 1 4 SEQ ID NO: 3280 attttgatgatgctggccc 8992 9011 SEQ ID NO: 4620 aaagaaacctatggcctta 13891 13910 1 4 SEQ ID NO: 3281 gaattatcttttaaacatt 8933 9012 SEQ ID NO: 4621 attcttaacgggattcctt 13890 13909 1 4 SEQ ID NO: 3282 ttaccaccagtttgaagtt 9041 9060 SEQ ID NO: 4622 taaagccattcaggtctct 12255 12274 1 4 SEQ ID NO: 3283 taagattaaatatttactt 9045 9064 SEQ ID NO: 4623 gacatgtttgaagggtgga 11667 11686 1 4 SEQ ID NO: 3284 aaagattactttgagaaat 9059 9078 SEQ ID NO: 4624 tgggctaaacgtttaattg 11653 11672 1 4 SEQ ID NO: 3285 aggaaatagttggattacc 9129 9148 SEQ ID NO: 4625 tattgaaatatgatttttt 9749 9768 1 4 SEQ ID NO: 3286 attttgatgatgctggccc 9132 9151 SEQ ID NO: 4626 agcttgtttgccagtgggc 11077 11096 1 4 SEQ ID NO: 3287 aggtttaaatggataattt 9262 9281 SEQ ID NO: 4627 aaagaaacctatggcctta 7977 7993 1 4 SEQ ID NO: 3288 cagaagctaagcaatgtcc 9279 9298 SEQ ID NO: 4628 attcttaacgggattcctt 9555 9574 1 4 SEQ ID NO: 3289 taagattaaatatttactt 9332 9351 SEQ ID NO: 4629 taaagccattcaggtctct 12838 12855 1 4 SEQ ID NO: 3290 aaagattactttgagaaat 9432 9451 SEQ ID NO: 4630 gacatgtttgaagggtgga 13608 13627 1 4 SEQ ID NO: 3291 aggaaatagttggattacc 9599 9818 SEQ ID NO: 4631 tgggctaaacgtttaattg 11085 11084 1 4 SEQ ID NO: 3292 attttgatgatgctggccc 9548 9557 SEQ ID NO: 4632 tattgaaatatgatttttt 9890 9709 1 4 SEQ ID NO: 3293 gaattatcttttaaacatt 9554 9673 SEQ ID NO: 4633 agcttgtttgccagtgggc 9280 9299 1 4 SEQ ID NO: 3294 ttaccaccagtttgaagtt 9887 9696 SEQ ID NO: 4634 ggacaaggcccagaatctg 10971 10990 1 4 SEQ ID NO: 3295 ttgcagtgtatcggacaga 9740 9759 SEQ ID NO: 4635 aaagaaacctatggcctta 12933 12952 1 4 SEQ ID NO: 3296 cattcagaaccagggaaga 9757 9776 SEQ ID NO: 4636 attcttaacgggattcctt 10279 10298 1 4 SEQ ID NO: 3297 acaaactgtttatagtccc 9817 9326 SEQ ID NO: 4637 taaagccattcaggtctct 11438 11457 1 4 SEQ ID NO: 3298 ggatttcaatacatttcac 9863 9832 SEQ ID NO: 4638 aattgttgaaagaaaaact 8810 8829 1 4 SEQ ID NO: 3299 ttgtagaaatgaaagtaaa 9890 9909 SEQ ID NO: 4639 ggacaaggcccagaatctg 9046 9065 1 4 SEQ ID NO: 3300 ctgaacagtgagctgcagt 9964 9933 SEQ ID NO: 4640 aaagaaacctatggcctta 6893 6912 1 4 SEQ ID NO: 3301 aatccaatctcctctttgc 9905 9984 SEQ ID NO: 4641 attcttaacgggattcctt 6892 6911 1 4 SEQ ID NO: 3302 atttgattttcaagcaaag 10019 10036 SEQ ID NO: 4642 taaagccattcaggtctct 13305 13324 1 4 SEQ ID NO: 3303 ttttgattttcaagcaaat 10177 10196 SEQ ID NO: 4643 gacatgtttgaagggtgga 11156 11205 1 4 SEQ ID NO: 3304 tgatttcaagcaaatgcag 10214 10233 SEQ ID NO: 4644 tgggctaaacgtttaattg 13772 13791 1 4 SEQ ID NO: 3305 atgctgtttgggaaattgg 10234 10253 SEQ ID NO: 4645 tattgaaatatgatttttt 1060 12099 1 4 SEQ ID NO: 3306 ttgtagaaatgaaagtaaa 10240 10259 SEQ ID NO: 4646 agcttgtttgccagtgggc 11668 11635 1 4 SEQ ID NO: 3307 ctgaacagtgagctgcagt 10309 10328 SEQ ID NO: 4647 tgaaatcattgaaaattta 10547 10566 1 4 SEQ ID NO: 3308 aatccaatctcctctttgc 10408 10427 SEQ ID NO: 4648 caggaggctttaagttgtc 13971 13990 1 4 SEQ ID NO: 3309 atttgattttcaagcaaag 10424 10443 SEQ ID NO: 4649 agcttgtttgccagtgggc 13216 13235 1 4 SEQ ID NO: 3310 ttttgattttcaagcaaat 10469 10488 SEQ ID NO: 4650 taaagccattcaggtctct 12591 12610 1 4 SEQ ID NO: 3311 tgatttcaagcaaatgcag 10583 10602 SEQ ID NO: 4651 aattgttgaaagaaaaact 12363 12382 1 4 SEQ ID NO: 3312 atgctgtttgggaaattgg 10918 10937 SEQ ID NO: 4652 taaagccattcaggtctct 5001 5020 1 4 SEQ ID NO: 3313 cagaagctaagcaatgtcc 10926 10945 SEQ ID NO: 4653 ttaatctttcataagtcaa 14002 14021 1 4 SEQ ID NO: 3314 taagattaaatatttactt 10983 11002 SEQ ID NO: 4654 ggacaaggcccagaatctg 8526 8545 1 4 SEQ ID NO: 3315 aaagattactttgagaaat 11004 11023 SEQ ID NO: 4655 attcttaacgggattcctt 12173 12192 1 4 SEQ ID NO: 3316 aggaaatagttggattacc 11264 11283 SEQ ID NO: 4656 aattgttgaaagaaaaact 11632 11651 1 4 SEQ ID NO: 3317 attttgatgatgctggccc 11268 11307 SEQ ID NO: 4657 agcttgtttgccagtgggc 9489 9508 1 4 SEQ ID NO: 3318 gaattatcttttaaacatt 11200 11309 SEQ ID NO: 4658 ttaatctttcataagtcaa 9047 9066 1 4 SEQ ID NO: 3319 ttaccaccagtttgaagtt 11315 11335 SEQ ID NO: 4659 taaagccattcaggtctct 11035 11054 1 4 SEQ ID NO: 3320 taagattaaatatttactt 11413 11432 SEQ ID NO: 4660 aattgttgaaagaaaaact 8873 8892 1 4 SEQ ID NO: 3321 aaagattactttgagaaat 11480 11499 SEQ ID NO: 4662 taaagccattcaggtctct 1206 12325 1 4 SEQ ID NO: 3322 aggaaatagttggattacc 11545 11584 SEQ ID NO: 4663 ttaatctttcataagtcaa 13723 13742 1 4 SEQ ID NO: 3323 attttgatgatgctggccc 11735 11754 SEQ ID NO: 4664 ggacaaggcccagaatctg 12102 12121 1 4 SEQ ID NO: 3324 gaattatcttttaaacatt 11796 11814 SEQ ID NO: 4665 attcttaacgggattcctt 13104 13123 1 4 SEQ ID NO: 3325 ttaccaccagtttgaagtt 11859 11876 SEQ ID NO: 4666 aattgttgaaagaaaaact 6254 6273 1 4 SEQ ID NO: 3326 ctgaaaaagttagtgaaag 11992 12011 SEQ ID NO: 4667 agcttgtttgccagtgggc 9220 9230 1 4 SEQ ID NO: 3327 tttttcccagacagtgtca 12050 12069 SEQ ID NO: 4668 ttaatctttcataagtcaa 12669 12666 1 4 SEQ ID NO: 3328 ttttcccagacagtgtcca 12143 12162 SEQ ID NO: 4669 agcttgtttgccagtgggc 9071 8000 1 4 SEQ ID NO: 3329 cattcagaaccagggaaga 12179 12198 SEQ ID NO: 4670 tgaaatcattgaaaattta 10088 10107 1 4 SEQ ID NO: 3330 acaaactgtttatagtccc 12219 12238 SEQ ID NO: 4671 caggaggctttaagttgtc 6993 7012 1 4 SEQ ID NO: 3331 ggatttcaatacatttcac 12428 13447 SEQ ID NO: 4672 agcttgtttgccagtgggc 13447 13466 1 4 SEQ ID NO: 3332 ttgtagaaatgaaagtaaa 12447 12466 SEQ ID NO: 4673 taaagccattcaggtctct 14056 14105 1 4 SEQ ID NO: 3333 ccaaaatttctctcggctt 12448 12487 SEQ ID NO: 4674 cttcatcgttcatttgagt 14085 14104 1 4 SEQ ID NO: 3334 caaattttctctgctaaaa 12477 12498 SEQ ID NO: 4675 cttatgggatttcctaagg 13392 13411 1 4 SEQ ID NO: 3335 tctgctggaaacaacaacg 12897 12916 SEQ ID NO: 4676 cttcatcgttcatttgagt 13641 13680 1 4 SEQ ID NO: 3336 cagaagctaagcaatgtcc 13039 13058 SEQ ID NO: 4677 ttccatcacaaatcctttg 14052 14071 1 4 SEQ ID NO: 3337 taagattaaatatttactt 13094 13114 SEQ ID NO: 4678 tctcaaagagttacaacag 15800 13819 1 4 SEQ ID NO: 3338 aaagattactttgagaaat 13151 13170 SEQ ID NO: 4679 tattgaaatatgatttttt 7032 7051 1 4 SEQ ID NO: 3339 aggaaatagttggattacc 13190 13209 SEQ ID NO: 4680 aattgttgaaagaaaaact 10168 10187 1 4 SEQ ID NO: 3340 attttgatgatgctggccc 13326 13346 SEQ ID NO: 4681 ggacaaggcccagaatctg 12407 12428 1 4 SEQ ID NO: 3341 gaattatcttttaaacatt 13377 13398 SEQ ID NO: 4682 aaagaaacctatggcctta 10737 10756 1 4 SEQ ID NO: 3342 ttaccaccagtttgaagtt 13451 13470 SEQ ID NO: 4683 attcttaacgggattcctt 11488 11307 1 4 SEQ ID NO: 3343 taagattaaatatttactt 13580 13579 SEQ ID NO: 4684 taaagccattcaggtctct 10103 10122 1 4 SEQ ID NO: 3344 aaagattactttgagaaat 13607 13626 SEQ ID NO: 4685 gacatgtttgaagggtgga 13873 13892 1 4 SEQ ID NO: 3345 aggaaatagttggattacc 13637 13858 SEQ ID NO: 4686 tgggctaaacgtttaattg 6436 6455 1 4 SEQ ID NO: 3346 attttgatgatgctggccc 13726 13745 SEQ ID NO: 4687 tattgaaatatgatttttt 9952 9971 1 4 SEQ ID NO: 3347 aggtttaaatggataattt 13773 13792 SEQ ID NO: 4688 agcttgtttgccagtgggc 10213 10232 1 4 SEQ ID NO: 3348 cagaagctaagcaatgtcc 13055 13974 SEQ ID NO: 4689 aaagaaacctatggcctta 7856 7875 1 4 SEQ ID NO: 3349 taagattaaatatttactt 14058 14077 SEQ ID NO: 4690 attcttaacgggattcctt 7230 4249 1 4 SEQ ID NO: 3350 aaagattactttgagaaat 4412 4431 SEQ ID NO: 4691 taaagccattcaggtctct 9603 9622 3 3 SEQ ID NO: 3351 aggaaatagttggattacc 4551 4570 SEQ ID NO: 4692 gacatgtttgaagggtgga 9063 9082 3 3 SEQ ID NO: 3352 attttgatgatgctggccc 33 52 SEQ ID NO: 4693 tgggctaaacgtttaattg 9236 9256 2 3 SEQ ID NO: 3353 gaattatcttttaaacatt 47 66 SEQ ID NO: 4694 tattgaaatatgatttttt 11185 11204 2 3 SEQ ID NO: 3354 ttaccaccagtttgaagtt 227 246 SEQ ID NO: 4695 agcttgtttgccagtgggc 8134 8153 2 3 SEQ ID NO: 3355 ttgcagtgtatcggacaga 291 310 SEQ ID NO: 4696 ggacaaggcccagaatctg 7465 7484 2 3 SEQ ID NO: 3356 cattcagaaccagggaaga 404 423 SEQ ID NO: 4697 aaagaaacctatggcctta 8977 8996 2 3 SEQ ID NO: 3357 acaaactgtttatagtccc 472 491 SEQ ID NO: 4698 attcttaacgggattcctt 11399 11418 2 3 SEQ ID NO: 3358 ggatttcaatacatttcac 582 601 SEQ ID NO: 4699 taaagccattcaggtctct 7050 7099 2 3 SEQ ID NO: 3359 ttgtagaaatgaaagtaaa 830 849 SEQ ID NO: 4700 taaagccattcaggtctct 8809 8825 2 3 SEQ ID NO: 3360 ctgaacagtgagctgcagt 864 884 SEQ ID NO: 4701 gacatgtttgaagggtgga 11354 11373 2 3 SEQ ID NO: 3361 aatccaatctcctctttgc 1087 1106 SEQ ID NO: 4702 tgggctaaacgtttaattg 12411 12430 2 3 SEQ ID NO: 3362 atttgattttcaagcaaag 1113 1132 SEQ ID NO: 4703 tattgaaatatgatttttt 13112 13131 2 3 SEQ ID NO: 3363 ttttgattttcaagcaaat 1176 1195 SEQ ID NO: 4704 agcttgtttgccagtgggc 3035 3054 2 3 SEQ ID NO: 3364 tgatttcaagcaaatgcag 1311 1330 SEQ ID NO: 4705 ggacaaggcccagaatctg 7856 7876 2 3 SEQ ID NO: 3365 atgctgtttgggaaattgg 1440 1459 SEQ ID NO: 4706 aaagaaacctatggcctta 9138 9157 2 3 SEQ ID NO: 3366 tgctgttttttggaaatgc 1500 1519 SEQ ID NO: 4707 attcttaacgggattcctt 10926 10947 2 3 SEQ ID NO: 3367 aaaaaaatacactggacgt 1575 1594 SEQ ID NO: 4708 aattgttgaaagaaaaact 7428 7447 2 3 SEQ ID NO: 3368 actggagcttagtaagggc 1627 1646 SEQ ID NO: 4709 ggacaaggcccagaatctg 5520 5539 2 3 SEQ ID NO: 3369 cttctggaaagagggtcat 1744 1783 SEQ ID NO: 4710 aaagaaacctatggcctta 10455 10474 2 3 SEQ ID NO: 3370 ggaaaagggtcatggaaat 1810 1829 SEQ ID NO: 4711 attcttaacgggattcctt 13734 13753 2 3 SEQ ID NO: 3371 gggcctgccccagatttct 1941 1960 SEQ ID NO: 4712 taaagccattcaggtctct 3552 3571 2 3 SEQ ID NO: 3372 ttctcagataggggaacac 1956 1976 SEQ ID NO: 4713 gacatgtttgaagggtgga 8275 8294 2 3 SEQ ID NO: 3373 gatgaaagaattaagggga 2084 2103 SEQ ID NO: 4714 tgggctaaacgtttaattg 10467 10486 2 3 SEQ ID NO: 3374 cttggactgtcaaataagt 2221 2240 SEQ ID NO: 4715 tattgaaatatgatttttt 8324 8343 2 3 SEQ ID NO: 3375 ggaaaagggtcatggaaat 2374 2393 SEQ ID NO: 4716 agcttgtttgccagtgggc 9668 9575 2 3 SEQ ID NO: 3376 aggaaatagttggattacc 2408 2427 SEQ ID NO: 4717 tgaaatcattgaaaattta 10037 10095 2 3 SEQ ID NO: 3377 attttgatgatgctggccc 2417 2436 SEQ ID NO: 4718 caggaggctttaagttgtc 4951 4970 2 3 SEQ ID NO: 3378 gaattatcttttaaacatt 2570 2569 SEQ ID NO: 4719 agcttgtttgccagtgggc 10020 10039 2 3 SEQ ID NO: 3379 ttaccaccagtttgaagtt 2583 2602 SEQ ID NO: 4720 taaagccattcaggtctct 12160 12179 2 3 SEQ ID NO: 3380 ctgaaaaagttagtgaaag 2765 2784 SEQ ID NO: 4721 aattgttgaaagaaaaact 11815 11834 2 3 SEQ ID NO: 3381 tttttcccagacagtgtca 3252 3271 SEQ ID NO: 4722 taaagccattcaggtctct 10903 10922 2 3 SEQ ID NO: 3382 ttttcccagacagtgtcca 3000 3667 SEQ ID NO: 4723 ttaatctttcataagtcaa 11450 11469 2 3 SEQ ID NO: 3383 cattcagaaccagggaaga 3681 3700 SEQ ID NO: 4724 ggacaaggcccagaatctg 12927 12946 2 3 SEQ ID NO: 3384 ttgcagtgtatcggacaga 3683 3702 SEQ ID NO: 4725 attcttaacgggattcctt 13785 13804 2 3 SEQ ID NO: 3385 cattcagaaccagggaaga 4104 4123 SEQ ID NO: 4726 aattgttgaaagaaaaact 9547 9566 2 3 SEQ ID NO: 3386 acaaactgtttatagtccc 4551 4570 SEQ ID NO: 4727 agcttgtttgccagtgggc 8247 8266 2 3 SEQ ID NO: 3387 ggatttcaatacatttcac 5135 5154 SEQ ID NO: 4728 ttaatctttcataagtcaa 13518 13537 2 3 SEQ ID NO: 3388 ttgtagaaatgaaagtaaa 5353 5372 SEQ ID NO: 4729 taaagccattcaggtctct 13874 13893 2 3 SEQ ID NO: 3389 ctgaacagtgagctgcagt 5420 5439 SEQ ID NO: 4730 aattgttgaaagaaaaact 8780 8799 2 3 SEQ ID NO: 3390 aatccaatctcctctttgc 5512 5331 SEQ ID NO: 4731 taaagccattcaggtctct 8794 8813 2 3 SEQ ID NO: 3391 atttgattttcaagcaaag 5848 5367 SEQ ID NO: 4732 ttaatctttcataagtcaa 11229 11248 2 3 SEQ ID NO: 3392 ttttgattttcaagcaaat 6285 5304 SEQ ID NO: 4733 ggacaaggcccagaatctg 13656 13676 2 3 SEQ ID NO: 3393 tgatttcaagcaaatgcag 6288 6307 SEQ ID NO: 4734 attcttaacgggattcctt 8538 8558 2 3 SEQ ID NO: 3394 atgctgtttgggaaattgg 6320 6339 SEQ ID NO: 4735 aattgttgaaagaaaaact 10756 10775 2 3 SEQ ID NO: 3395 tgctgttttttggaaatgc 6488 6507 SEQ ID NO: 4736 agcttgtttgccagtgggc 7162 7181 2 3 SEQ ID NO: 3396 aaaaaaatacactggacgt 6506 6525 SEQ ID NO: 4737 ttaatctttcataagtcaa 11324 11343 2 3 SEQ ID NO: 3397 ctgaaaaagttagtgaaag 6696 6715 SEQ ID NO: 4738 agcttgtttgccagtgggc 13441 13460 2 3 SEQ ID NO: 3398 tttttcccagacagtgtca 7060 7079 SEQ ID NO: 4739 tgaaatcattgaaaattta 12971 12990 2 3 SEQ ID NO: 3399 ttttcccagacagtgtcca 7356 7375 SEQ ID NO: 4740 caggaggctttaagttgtc 12660 12679 2 3 SEQ ID NO: 3400 cattcagaaccagggaaga 7753 7772 SEQ ID NO: 4741 agcttgtttgccagtgggc 13276 13295 2 3 SEQ ID NO: 3401 acaaactgtttatagtccc 8080 8069 SEQ ID NO: 4742 taaagccattcaggtctct 9481 9500 2 3 SEQ ID NO: 3402 ggatttcaatacatttcac 9408 9424 SEQ ID NO: 4743 cttcatcgttcatttgagt 14083 14102 2 3 SEQ ID NO: 3403 ttgtagaaatgaaagtaaa 8 27 SEQ ID NO: 4744 cttatgggatttcctaagg 11571 11590 1 3 SEQ ID NO: 3404 ccaaaatttctctcggctt 25 44 SEQ ID NO: 4745 cttcatcgttcatttgagt 12078 12697 1 3 SEQ ID NO: 3405 caaattttctctgctaaaa 47 86 SEQ ID NO: 4746 ttccatcacaaatcctttg 1225 1244 1 3 SEQ ID NO: 3406 tctgctggaaacaacaacg 50 69 SEQ ID NO: 4747 tctcaaagagttacaacag 3752 3771 1 3 SEQ ID NO: 3407 cagaagctaagcaatgtcc 72 91 SEQ ID NO: 4748 tattgaaatatgatttttt 1383 1382 1 3 SEQ ID NO: 3408 taagattaaatatttactt 89 108 SEQ ID NO: 4749 aattgttgaaagaaaaact 2692 2701 1 3 SEQ ID NO: 3409 aaagattactttgagaaat 104 123 SEQ ID NO: 4750 ggacaaggcccagaatctg 1086 1107 1 3 SEQ ID NO: 3410 aggaaatagttggattacc 200 210 SEQ ID NO: 4751 aaagaaacctatggcctta 8309 8328 1 3 SEQ ID NO: 3411 attttgatgatgctggccc 237 256 SEQ ID NO: 4752 attcttaacgggattcctt 7085 7104 1 3 SEQ ID NO: 3412 gaattatcttttaaacatt 253 272 SEQ ID NO: 4753 taaagccattcaggtctct 10067 10086 1 3 SEQ ID NO: 3413 ttaccaccagtttgaagtt 297 316 SEQ ID NO: 4754 gacatgtttgaagggtgga 8610 8629 1 3 SEQ ID NO: 3414 taagattaaatatttactt 316 335 SEQ ID NO: 4755 tgggctaaacgtttaattg 13324 13345 1 3 SEQ ID NO: 3415 aaagattactttgagaaat 333 352 SEQ ID NO: 4756 tattgaaatatgatttttt 10704 10723 1 3 SEQ ID NO: 3416 aggaaatagttggattacc 343 362 SEQ ID NO: 4757 agcttgtttgccagtgggc 4748 4767 1 3 SEQ ID NO: 3417 attttgatgatgctggccc 348 367 SEQ ID NO: 4758 aaagaaacctatggcctta 1289 1305 1 3 SEQ ID NO: 3418 aggtttaaatggataattt 373 392 SEQ ID NO: 4759 attcttaacgggattcctt 1343 1382 1 3 SEQ ID NO: 3419 cagaagctaagcaatgtcc 383 402 SEQ ID NO: 4760 taaagccattcaggtctct 5112 5131 1 3 SEQ ID NO: 3420 taagattaaatatttactt 385 404 SEQ ID NO: 4761 gacatgtttgaagggtgga 2698 2715 1 3 SEQ ID NO: 3421 aaagattactttgagaaat 399 418 SEQ ID NO: 4762 tgggctaaacgtttaattg 1539 1588 1 3 SEQ ID NO: 3422 aggaaatagttggattacc 404 423 SEQ ID NO: 4763 tattgaaatatgatttttt 5230 5249 1 3 SEQ ID NO: 3423 attttgatgatgctggccc 427 446 SEQ ID NO: 4764 agcttgtttgccagtgggc 3533 3652 1 3 SEQ ID NO: 3424 gaattatcttttaaacatt 430 449 SEQ ID NO: 4765 ggacaaggcccagaatctg 2207 2226 1 3 SEQ ID NO: 3425 ttaccaccagtttgaagtt 431 450 SEQ ID NO: 4766 aaagaaacctatggcctta 9727 9746 1 3 SEQ ID NO: 3426 ttgcagtgtatcggacaga 451 470 SEQ ID NO: 4767 attcttaacgggattcctt 9074 9093 1 3 SEQ ID NO: 3427 cattcagaaccagggaaga 453 472 SEQ ID NO: 4768 taaagccattcaggtctct 5647 5666 1 3 SEQ ID NO: 3428 acaaactgtttatagtccc 483 503 SEQ ID NO: 4769 taaagccattcaggtctct 3004 3023 1 3 SEQ ID NO: 3429 ggatttcaatacatttcac 484 503 SEQ ID NO: 4770 gacatgtttgaagggtgga 3003 3022 1 3 SEQ ID NO: 3430 ttgtagaaatgaaagtaaa 490 509 SEQ ID NO: 4771 tgggctaaacgtttaattg 1293 1312 1 3 SEQ ID NO: 3431 ctgaacagtgagctgcagt 507 526 SEQ ID NO: 4772 tattgaaatatgatttttt 7489 7508 1 3 SEQ ID NO: 3432 aatccaatctcctctttgc 526 545 SEQ ID NO: 4773 agcttgtttgccagtgggc 13821 13840 1 3 SEQ ID NO: 3433 atttgattttcaagcaaag 628 5547 SEQ ID NO: 4774 ggacaaggcccagaatctg 7572 7591 1 3 SEQ ID NO: 3434 ttttgattttcaagcaaat 555 574 SEQ ID NO: 4775 aaagaaacctatggcctta 4852 4871 1 3 SEQ ID NO: 3435 tgatttcaagcaaatgcag 575 594 SEQ ID NO: 4776 attcttaacgggattcctt 7977 7995 1 3 SEQ ID NO: 3436 atgctgtttgggaaattgg 576 595 SEQ ID NO: 4777 taaagccattcaggtctct 7976 7995 1 3 SEQ ID NO: 3437 ttgtagaaatgaaagtaaa 576 597 SEQ ID NO: 4778 tattgaaatatgatttttt 7908 7927 1 3 SEQ ID NO: 3438 ctgaacagtgagctgcagt 581 600 SEQ ID NO: 4779 agcttgtttgccagtgggc 5298 6317 1 3 SEQ ID NO: 3439 aatccaatctcctctttgc 582 601 SEQ ID NO: 4780 ggacaaggcccagaatctg 6297 8316 1 3 SEQ ID NO: 3440 atttgattttcaagcaaag 597 616 SEQ ID NO: 4781 aaagaaacctatggcctta 7413 7432 1 3 SEQ ID NO: 3441 ttttgattttcaagcaaat 615 634 SEQ ID NO: 4782 taaagccattcaggtctct 10066 10085 1 3 SEQ ID NO: 3442 tgatttcaagcaaatgcag 629 648 SEQ ID NO: 4783 taaagccattcaggtctct 8079 8098 1 3 SEQ ID NO: 3443 atgctgtttgggaaattgg 647 666 SEQ ID NO: 4784 gacatgtttgaagggtgga 8775 8794 1 3 SEQ ID NO: 3444 cagaagctaagcaatgtcc 663 682 SEQ ID NO: 4785 tgggctaaacgtttaattg 2928 2947 1 3 SEQ ID NO: 3445 taagattaaatatttactt 670 689 SEQ ID NO: 4786 tgggctaaacgtttaattg 4540 4559 1 3 SEQ ID NO: 3446 aaagattactttgagaaat 660 699 SEQ ID NO: 4787 attcttaacgggattcctt 10580 10599 1 3 SEQ ID NO: 3447 aggaaatagttggattacc 665 704 SEQ ID NO: 4788 aattgttgaaagaaaaact 2491 2510 1 3 SEQ ID NO: 3448 attttgatgatgctggccc 687 706 SEQ ID NO: 4789 agcttgtttgccagtgggc 11177 11198 1 3 SEQ ID NO: 3449 gaattatcttttaaacatt 698 717 SEQ ID NO: 4790 ggacaaggcccagaatctg 5964 5983 1 3 SEQ ID NO: 3450 ttaccaccagtttgaagtt 699 718 SEQ ID NO: 4791 aaagaaacctatggcctta 3515 3534 1 3 SEQ ID NO: 3451 taagattaaatatttactt 700 719 SEQ ID NO: 4792 attcttaacgggattcctt 3476 2495 1 3 SEQ ID NO: 3452 aaagattactttgagaaat 702 721 SEQ ID NO: 4793 taaagccattcaggtctct 13364 13383 1 3 SEQ ID NO: 3453 aggaaatagttggattacc 703 722 SEQ ID NO: 4794 taaagccattcaggtctct 1809 1828 1 3 SEQ ID NO: 3454 attttgatgatgctggccc 777 798 SEQ ID NO: 4795 gacatgtttgaagggtgga 3019 3038 1 3 SEQ ID NO: 3455 gaattatcttttaaacatt 776 797 SEQ ID NO: 4796 tgggctaaacgtttaattg 3018 3037 1 3 SEQ ID NO: 3456 ttaccaccagtttgaagtt 783 802 SEQ ID NO: 4797 tattgaaatatgatttttt 12668 12687 1 3 SEQ ID NO: 3457 ctgaaaaagttagtgaaag 823 842 SEQ ID NO: 4798 agcttgtttgccagtgggc 4819 4838 1 3 SEQ ID NO: 3458 tttttcccagacagtgtca 885 884 SEQ ID NO: 4799 ggacaaggcccagaatctg 8339 9358 1 3 SEQ ID NO: 3459 ttttcccagacagtgtcca 902 921 SEQ ID NO: 4800 aaagaaacctatggcctta 13465 13484 1 3 SEQ ID NO: 3460 cattcagaaccagggaaga 903 922 SEQ ID NO: 4801 attcttaacgggattcctt 13464 13483 1 3 SEQ ID NO: 3461 acaaactgtttatagtccc 908 927 SEQ ID NO: 4802 taaagccattcaggtctct 4292 4311 1 3 SEQ ID NO: 3462 ggatttcaatacatttcac 909 928 SEQ ID NO: 4803 tattgaaatatgatttttt 1075 1094 1 3 SEQ ID NO: 3463 ttgtagaaatgaaagtaaa 933 952 SEQ ID NO: 4804 agcttgtttgccagtgggc 5794 6813 1 3 SEQ ID NO: 3464 ccaaaatttctctcggctt 934 953 SEQ ID NO: 4805 ggacaaggcccagaatctg 1246 1265 1 3 SEQ ID NO: 3465 caaattttctctgctaaaa 954 973 SEQ ID NO: 4806 ggacaaggcccagaatctg 4592 4611 1 3 SEQ ID NO: 3466 tctgctggaaacaacaacg 955 974 SEQ ID NO: 4807 attcttaacgggattcctt 4591 4610 1 3 SEQ ID NO: 3467 ttgtagaaatgaaagtaaa 956 975 SEQ ID NO: 4808 aattgttgaaagaaaaact 5099 5116 1 3 SEQ ID NO: 3468 ccaaaatttctctcggctt 978 997 SEQ ID NO: 4809 agcttgtttgccagtgggc 7987 8006 1 3 SEQ ID NO: 3469 caaattttctctgctaaaa 1008 1027 SEQ ID NO: 4810 ttaatctttcataagtcaa 8372 8391 1 3 SEQ ID NO: 3470 tctgctggaaacaacaacg 1012 1031 SEQ ID NO: 4811 taaagccattcaggtctct 11167 11186 1 3 SEQ ID NO: 3471 cagaagctaagcaatgtcc 1024 1043 SEQ ID NO: 4812 aattgttgaaagaaaaact 10337 10366 1 3 SEQ ID NO: 3472 taagattaaatatttactt 1045 1065 SEQ ID NO: 4813 taaagccattcaggtctct 10380 10399 1 3 SEQ ID NO: 3473 aaagattactttgagaaat 1050 1069 SEQ ID NO: 4814 ttaatctttcataagtcaa 4876 4695 1 3 SEQ ID NO: 3474 aggaaatagttggattacc 1087 1106 SEQ ID NO: 4815 ggacaaggcccagaatctg 5880 5899 1 3 SEQ ID NO: 3475 attttgatgatgctggccc 1113 1132 SEQ ID NO: 4816 attcttaacgggattcctt 7112 7131 1 3 SEQ ID NO: 3476 gaattatcttttaaacatt 1114 1133 SEQ ID NO: 4817 aattgttgaaagaaaaact 11052 11071 1 3 SEQ ID NO: 3477 ttaccaccagtttgaagtt 1130 1149 SEQ ID NO: 4818 agcttgtttgccagtgggc 12260 12279 1 3 SEQ ID NO: 3478 taagattaaatatttactt 1156 1175 SEQ ID NO: 4819 ttaatctttcataagtcaa 6326 6345 1 3 SEQ ID NO: 3479 aaagattactttgagaaat 1176 1196 SEQ ID NO: 4820 agcttgtttgccagtgggc 1387 1386 1 3 SEQ ID NO: 3480 aggaaatagttggattacc 1198 1217 SEQ ID NO: 4821 tgaaatcattgaaaattta 7112 7131 1 3 SEQ ID NO: 3481 attttgatgatgctggccc 1200 1219 SEQ ID NO: 4822 caggaggctttaagttgtc 12297 12316 1 3 SEQ ID NO: 3482 aggtttaaatggataattt 1212 1231 SEQ ID NO: 4823 agcttgtttgccagtgggc 12948 12965 1 3 SEQ ID NO: 3483 cagaagctaagcaatgtcc 1215 1234 SEQ ID NO: 4824 taaagccattcaggtctct 12279 12298 1 3 SEQ ID NO: 3484 taagattaaatatttactt 1216 1235 SEQ ID NO: 4825 cttcatcgttcatttgagt 8528 8547 1 3 SEQ ID NO: 3485 aaagattactttgagaaat 1231 1250 SEQ ID NO: 4826 cttatgggatttcctaagg 4733 4752 1 3 SEQ ID NO: 3486 aggaaatagttggattacc 1267 1286 SEQ ID NO: 4827 cttcatcgttcatttgagt 5001 5020 1 3 SEQ ID NO: 3487 attttgatgatgctggccc 1296 1315 SEQ ID NO: 4828 ttccatcacaaatcctttg 7239 7258 1 3 SEQ ID NO: 3488 gaattatcttttaaacatt 1385 1404 SEQ ID NO: 4829 tctcaaagagttacaacag 12323 12342 1 3 SEQ ID NO: 3489 ttaccaccagtttgaagtt 1388 1407 SEQ ID NO: 4830 tattgaaatatgatttttt 13051 13070 1 3 SEQ ID NO: 3490 ttgcagtgtatcggacaga 1415 1434 SEQ ID NO: 4831 aattgttgaaagaaaaact 5718 5737 1 3 SEQ ID NO: 3491 cattcagaaccagggaaga 1478 1497 SEQ ID NO: 4832 ggacaaggcccagaatctg 3936 3955 1 3 SEQ ID NO: 3492 acaaactgtttatagtccc 1499 1518 SEQ ID NO: 4833 aaagaaacctatggcctta 4660 4679 1 3 SEQ ID NO: 3493 ggatttcaatacatttcac 1500 1519 SEQ ID NO: 4834 attcttaacgggattcctt 7664 7883 1 3 SEQ ID NO: 3494 ttgtagaaatgaaagtaaa 1505 1524 SEQ ID NO: 4835 taaagccattcaggtctct 12287 12306 1 3 SEQ ID NO: 3495 ctgaacagtgagctgcagt 1565 1584 SEQ ID NO: 4836 gacatgtttgaagggtgga 6353 6372 1 3 SEQ ID NO: 3496 aatccaatctcctctttgc 1575 1594 SEQ ID NO: 4837 tgggctaaacgtttaattg 7161 7180 1 3 SEQ ID NO: 3497 atttgattttcaagcaaag 1582 1801 SEQ ID NO: 4838 tattgaaatatgatttttt 6436 8455 1 3 SEQ ID NO: 3498 ttttgattttcaagcaaat 1609 1826 SEQ ID NO: 4839 agcttgtttgccagtgggc 9962 9981 1 3 SEQ ID NO: 3499 tgatttcaagcaaatgcag 1615 1834 SEQ ID NO: 4840 aaagaaacctatggcctta 8400 8419 1 3 SEQ ID NO: 3500 atgctgtttgggaaattgg 1625 1844 SEQ ID NO: 4841 attcttaacgggattcctt 11654 11673 1 3 SEQ ID NO: 3501 tgctgttttttggaaatgc 1627 1845 SEQ ID NO: 4842 taaagccattcaggtctct 1920 1939 1 3 SEQ ID NO: 3502 aaaaaaatacactggacgt 1663 1682 SEQ ID NO: 4843 gacatgtttgaagggtgga 4429 4448 1 3 SEQ ID NO: 3503 actggagcttagtaagggc 1672 1691 SEQ ID NO: 4844 tgggctaaacgtttaattg 10883 10902 1 3 SEQ ID NO: 3504 cttctggaaagagggtcat 1673 1692 SEQ ID NO: 4845 tattgaaatatgatttttt 13999 14016 1 3 SEQ ID NO: 3505 ggaaaagggtcatggaaat 1685 1704 SEQ ID NO: 4846 agcttgtttgccagtgggc 11173 11192 1 3 SEQ ID NO: 3506 gggcctgccccagatttct 1688 1707 SEQ ID NO: 4847 ggacaaggcccagaatctg 5518 5537 1 3 SEQ ID NO: 3507 ttctcagataggggaacac 1731 1750 SEQ ID NO: 4848 aaagaaacctatggcctta 4542 4561 1 3 SEQ ID NO: 3508 gatgaaagaattaagggga 1734 1753 SEQ ID NO: 4849 attcttaacgggattcctt 7242 7261 1 3 SEQ ID NO: 3509 cttggactgtcaaataagt 1737 1756 SEQ ID NO: 4850 taaagccattcaggtctct 4290 4309 1 3 SEQ ID NO: 3510 ggaaaagggtcatggaaat 1750 1789 SEQ ID NO: 4851 taaagccattcaggtctct 2567 2566 1 3 SEQ ID NO: 3511 aggaaatagttggattacc 1752 1771 SEQ ID NO: 4852 gacatgtttgaagggtgga 11307 11326 1 3 SEQ ID NO: 3512 attttgatgatgctggccc 1810 1829 SEQ ID NO: 4853 tgggctaaacgtttaattg 9046 9065 1 3 SEQ ID NO: 3513 gaattatcttttaaacatt 1824 1843 SEQ ID NO: 4854 tattgaaatatgatttttt 7148 7167 1 3 SEQ ID NO: 3514 ttaccaccagtttgaagtt 1825 1844 SEQ ID NO: 4855 agcttgtttgccagtgggc 7147 7155 1 3 SEQ ID NO: 3515 ctgaaaaagttagtgaaag 1831 1850 SEQ ID NO: 4856 ggacaaggcccagaatctg 6589 6608 1 3 SEQ ID NO: 3516 tttttcccagacagtgtca 1836 1855 SEQ ID NO: 4857 aaagaaacctatggcctta 2760 2779 1 3 SEQ ID NO: 3517 ttttcccagacagtgtcca 1842 1861 SEQ ID NO: 4858 attcttaacgggattcctt 4542 4561 1 3 SEQ ID NO: 3518 cattcagaaccagggaaga 1877 1896 SEQ ID NO: 4859 aattgttgaaagaaaaact 5334 5353 1 3 SEQ ID NO: 3519 ttgcagtgtatcggacaga 1883 1902 SEQ ID NO: 4860 ggacaaggcccagaatctg 12677 12698 1 3 SEQ ID NO: 3520 cattcagaaccagggaaga 1887 1900 SEQ ID NO: 4861 aaagaaacctatggcctta 8909 5928 1 3 SEQ ID NO: 3521 acaaactgtttatagtccc 1892 1911 SEQ ID NO: 4862 attcttaacgggattcctt 8998 9017 1 3 SEQ ID NO: 3522 ggatttcaatacatttcac 1896 1915 SEQ ID NO: 4863 taaagccattcaggtctct 7631 7850 1 3 SEQ ID NO: 3523 ttgtagaaatgaaagtaaa 1900 1919 SEQ ID NO: 4864 gacatgtttgaagggtgga 12745 12764 1 3 SEQ ID NO: 3524 ctgaacagtgagctgcagt 1901 1920 SEQ ID NO: 4865 tgggctaaacgtttaattg 12276 12296 1 3 SEQ ID NO: 3525 aatccaatctcctctttgc 1908 1927 SEQ ID NO: 4866 tattgaaatatgatttttt 9352 9371 1 3 SEQ ID NO: 3526 atttgattttcaagcaaag 1934 1953 SEQ ID NO: 4867 agcttgtttgccagtgggc 9400 9419 1 3 SEQ ID NO: 3527 ttttgattttcaagcaaat 1941 1960 SEQ ID NO: 4868 tgaaatcattgaaaattta 1986 2005 1 3 SEQ ID NO: 3528 tgatttcaagcaaatgcag 1949 1968 SEQ ID NO: 4869 caggaggctttaagttgtc 7915 7934 1 3 SEQ ID NO: 3529 atgctgtttgggaaattgg 1955 1975 SEQ ID NO: 4870 agcttgtttgccagtgggc 1978 1997 1 3 SEQ ID NO: 3530 tgctgttttttggaaatgc 1980 1999 SEQ ID NO: 4871 taaagccattcaggtctct 12350 12369 1 3 SEQ ID NO: 3531 aaaaaaatacactggacgt 1997 2016 SEQ ID NO: 4872 aattgttgaaagaaaaact 7036 7055 1 3 SEQ ID NO: 3532 ctgaaaaagttagtgaaag 2029 2048 SEQ ID NO: 4873 taaagccattcaggtctct 6976 6997 1 3 SEQ ID NO: 3533 tttttcccagacagtgtca 2031 2050 SEQ ID NO: 4874 ttaatctttcataagtcaa 10284 10303 1 3 SEQ ID NO: 3534 ttttcccagacagtgtcca 2079 2098 SEQ ID NO: 4875 ggacaaggcccagaatctg 5457 5476 1 3 SEQ ID NO: 3535 cattcagaaccagggaaga 2086 2102 SEQ ID NO: 4876 attcttaacgggattcctt 4900 4919 1 3 SEQ ID NO: 3536 acaaactgtttatagtccc 2084 2103 SEQ ID NO: 4877 aattgttgaaagaaaaact 5287 5305 1 3 SEQ ID NO: 3537 ggatttcaatacatttcac 2101 2120 SEQ ID NO: 4878 agcttgtttgccagtgggc 3461 3480 1 3 SEQ ID NO: 3538 ttgtagaaatgaaagtaaa 2158 2177 SEQ ID NO: 4879 ttaatctttcataagtcaa 10932 10951 1 3 SEQ ID NO: 3539 ccaaaatttctctcggctt 2162 2181 SEQ ID NO: 4880 taaagccattcaggtctct 12265 12284 1 3 SEQ ID NO: 3540 caaattttctctgctaaaa 2191 2210 SEQ ID NO: 4881 aattgttgaaagaaaaact 7850 7869 1 3 SEQ ID NO: 3541 tctgctggaaacaacaacg 2193 2212 SEQ ID NO: 4882 taaagccattcaggtctct 4086 4105 1 3 SEQ ID NO: 3542 cagaagctaagcaatgtcc 2204 2223 SEQ ID NO: 4883 attcttaacgggattcctt 12956 12977 1 3 SEQ ID NO: 3543 taagattaaatatttactt 2209 2228 SEQ ID NO: 4884 taaagccattcaggtctct 13521 13540 1 3 SEQ ID NO: 3544 aaagattactttgagaaat 2210 2229 SEQ ID NO: 4885 gacatgtttgaagggtgga 13520 13530 1 3 SEQ ID NO: 3545 aggaaatagttggattacc 2215 2234 SEQ ID NO: 4886 tgggctaaacgtttaattg 4539 4558 1 3 SEQ ID NO: 3546 attttgatgatgctggccc 2220 2239 SEQ ID NO: 4887 tattgaaatatgatttttt 3785 3804 1 3 SEQ ID NO: 3547 gaattatcttttaaacatt 2226 2245 SEQ ID NO: 4888 agcttgtttgccagtgggc 7796 7817 1 3 SEQ ID NO: 3548 ttaccaccagtttgaagtt 2229 2248 SEQ ID NO: 4889 tgaaatcattgaaaattta 6730 5749 1 3 SEQ ID NO: 3549 taagattaaatatttactt 2247 2266 SEQ ID NO: 4890 caggaggctttaagttgtc 6723 6742 1 3 SEQ ID NO: 3550 aaagattactttgagaaat 2331 2350 SEQ ID NO: 4891 agcttgtttgccagtgggc 2576 2696 1 3 SEQ ID NO: 3551 aggaaatagttggattacc 2337 2366 SEQ ID NO: 4892 taaagccattcaggtctct 6280 6299 1 3 SEQ ID NO: 3552 attttgatgatgctggccc 2357 2376 SEQ ID NO: 4893 aattgttgaaagaaaaact 4262 4281 1 3 SEQ ID NO: 3553 aggtttaaatggataattt 2365 2385 SEQ ID NO: 4894 taaagccattcaggtctct 9954 9973 1 3 SEQ ID NO: 3554 cagaagctaagcaatgtcc 2367 2398 SEQ ID NO: 4895 ttaatctttcataagtcaa 10809 10525 1 3 SEQ ID NO: 3555 taagattaaatatttactt 2370 2389 SEQ ID NO: 4896 ggacaaggcccagaatctg 13067 13086 1 3 SEQ ID NO: 3556 aaagattactttgagaaat 2374 2393 SEQ ID NO: 4897 attcttaacgggattcctt 7981 8000 1 3 SEQ ID NO: 3557 aggaaatagttggattacc 2385 2404 SEQ ID NO: 4898 aattgttgaaagaaaaact 9542 9501 1 3 SEQ ID NO: 3558 attttgatgatgctggccc 2386 2405 SEQ ID NO: 4899 agcttgtttgccagtgggc 9541 9550 1 3 SEQ ID NO: 3559 gaattatcttttaaacatt 2387 2406 SEQ ID NO: 4900 ttaatctttcataagtcaa 9540 9560 1 3 SEQ ID NO: 3560 ttaccaccagtttgaagtt 2401 2420 SEQ ID NO: 4901 taaagccattcaggtctct 6874 6893 1 3 SEQ ID NO: 3561 ttgcagtgtatcggacaga 1408 2427 SEQ ID NO: 4902 aattgttgaaagaaaaact 8248 8267 1 3 SEQ ID NO: 3562 cattcagaaccagggaaga 1409 2428 SEQ ID NO: 4903 taaagccattcaggtctct 8247 8266 1 3 SEQ ID NO: 3563 acaaactgtttatagtccc 2416 2435 SEQ ID NO: 4904 agcttgtttgccagtgggc 3598 3707 1 3 SEQ ID NO: 3564 ggatttcaatacatttcac 2417 2436 SEQ ID NO: 4905 ggacaaggcccagaatctg 3887 3706 1 3 SEQ ID NO: 3565 ttgtagaaatgaaagtaaa 2438 2457 SEQ ID NO: 4906 aaagaaacctatggcctta 8571 8590 1 3 SEQ ID NO: 3566 ctgaacagtgagctgcagt 2439 2458 SEQ ID NO: 4907 attcttaacgggattcctt 8570 8559 1 3 SEQ ID NO: 3567 aatccaatctcctctttgc 2465 2474 SEQ ID NO: 4908 taaagccattcaggtctct 12527 12545 1 3 SEQ ID NO: 3568 atttgattttcaagcaaag 2464 2483 SEQ ID NO: 4909 tattgaaatatgatttttt 2734 2763 1 3 SEQ ID NO: 3569 ttttgattttcaagcaaat 2469 2488 SEQ ID NO: 4910 agcttgtttgccagtgggc 10771 10790 1 3 SEQ ID NO: 3570 tgatttcaagcaaatgcag 2479 2468 SEQ ID NO: 4911 ggacaaggcccagaatctg 3538 3887 1 3 SEQ ID NO: 3571 atgctgtttgggaaattgg 3483 2502 SEQ ID NO: 4912 ggacaaggcccagaatctg 2672 2691 1 3 SEQ ID NO: 3572 ttgtagaaatgaaagtaaa 2601 2520 SEQ ID NO: 4913 attcttaacgggattcctt 7939 7958 1 3 SEQ ID NO: 3573 ctgaacagtgagctgcagt 2551 2580 SEQ ID NO: 4914 aattgttgaaagaaaaact 3514 3533 1 3 SEQ ID NO: 3574 aatccaatctcctctttgc 2582 2601 SEQ ID NO: 4915 agcttgtttgccagtgggc 3836 3854 1 3 SEQ ID NO: 3575 atttgattttcaagcaaag 2590 2509 SEQ ID NO: 4916 ttaatctttcataagtcaa 6107 5128 1 3 SEQ ID NO: 3576 ttttgattttcaagcaaat 2605 2624 SEQ ID NO: 4917 taaagccattcaggtctct 10718 10737 1 3 SEQ ID NO: 3577 tgatttcaagcaaatgcag 2606 2627 SEQ ID NO: 4918 aattgttgaaagaaaaact 12281 12300 1 3 SEQ ID NO: 3578 atgctgtttgggaaattgg 2618 2637 SEQ ID NO: 4919 taaagccattcaggtctct 5737 5759 1 3 SEQ ID NO: 3579 cagaagctaagcaatgtcc 2621 2640 SEQ ID NO: 4920 ttaatctttcataagtcaa 5383 5402 1 3 SEQ ID NO: 3580 taagattaaatatttactt 2692 2711 SEQ ID NO: 4921 ggacaaggcccagaatctg 5080 6099 1 3 SEQ ID NO: 3581 cttggactgtcaaataagt 2698 2717 SEQ ID NO: 4922 attcttaacgggattcctt 3059 076 1 3 SEQ ID NO: 3582 ggaaaagggtcatggaaat 2710 2729 SEQ ID NO: 4923 aattgttgaaagaaaaact 4106 4125 1 3 SEQ ID NO: 3583 aggaaatagttggattacc 2765 2734 SEQ ID NO: 4924 agcttgtttgccagtgggc 11236 11255 1 3 SEQ ID NO: 3584 attttgatgatgctggccc 2767 2785 SEQ ID NO: 4925 ttaatctttcataagtcaa 4385 4404 1 3 SEQ ID NO: 3585 gaattatcttttaaacatt 2794 2813 SEQ ID NO: 4926 agcttgtttgccagtgggc 12341 12380 1 3 SEQ ID NO: 3586 ttaccaccagtttgaagtt 2808 2827 SEQ ID NO: 4927 tgaaatcattgaaaattta 4179 4198 1 3 SEQ ID NO: 3587 ctgaaaaagttagtgaaag 2834 2853 SEQ ID NO: 4928 caggaggctttaagttgtc 12562 12581 1 3 SEQ ID NO: 3588 tttttcccagacagtgtca 2841 2850 SEQ ID NO: 4929 agcttgtttgccagtgggc 4807 4828 1 3 SEQ ID NO: 3589 ttttcccagacagtgtcca 2843 2882 SEQ ID NO: 4930 taaagccattcaggtctct 7639 7665 1 3 SEQ ID NO: 3590 cattcagaaccagggaaga 2869 2888 SEQ ID NO: 4931 cttcatcgttcatttgagt 7951 7970 1 3 SEQ ID NO: 3591 ttgcagtgtatcggacaga 2879 2898 SEQ ID NO: 4932 cttatgggatttcctaagg 6269 6288 1 3 SEQ ID NO: 3592 cattcagaaccagggaaga 2908 2927 SEQ ID NO: 4933 cttcatcgttcatttgagt 13214 13233 1 3 SEQ ID NO: 3593 acaaactgtttatagtccc 2934 2953 SEQ ID NO: 4934 ttccatcacaaatcctttg 10191 10210 1 3 SEQ ID NO: 3594 ggatttcaatacatttcac 2939 2958 SEQ ID NO: 4935 tctcaaagagttacaacag 11889 11908 1 3 SEQ ID NO: 3595 ttgtagaaatgaaagtaaa 2940 2959 SEQ ID NO: 4936 tattgaaatatgatttttt 7106 7125 1 3 SEQ ID NO: 3596 ctgaacagtgagctgcagt 2954 2983 SEQ ID NO: 4937 aattgttgaaagaaaaact 3205 3224 1 3 SEQ ID NO: 3597 aatccaatctcctctttgc 2987 3006 SEQ ID NO: 4938 ggacaaggcccagaatctg 12031 12050 1 3 SEQ ID NO: 3598 atttgattttcaagcaaag 2989 3006 SEQ ID NO: 4939 aaagaaacctatggcctta 8341 8360 1 3 SEQ ID NO: 3599 ttttgattttcaagcaaat 3043 3062 SEQ ID NO: 4940 attcttaacgggattcctt 8440 6459 1 3 SEQ ID NO: 3600 tgatttcaagcaaatgcag 3074 3093 SEQ ID NO: 4941 taaagccattcaggtctct 3192 3211 1 3 SEQ ID NO: 3601 atgctgtttgggaaattgg 3101 3120 SEQ ID NO: 4942 gacatgtttgaagggtgga 12259 12278 1 3 SEQ ID NO: 3602 tgctgttttttggaaatgc 3120 3139 SEQ ID NO: 4943 tgggctaaacgtttaattg 5144 5163 1 3 SEQ ID NO: 3603 aaaaaaatacactggacgt 3175 3194 SEQ ID NO: 4944 tattgaaatatgatttttt 6549 6668 1 3 SEQ ID NO: 3604 ctgaaaaagttagtgaaag 3202 3221 SEQ ID NO: 4945 agcttgtttgccagtgggc 13217 13236 1 3 SEQ ID NO: 3605 tttttcccagacagtgtca 3222 3241 SEQ ID NO: 4946 aaagaaacctatggcctta 9630 9549 1 3 SEQ ID NO: 3606 ttttcccagacagtgtcca 3225 3244 SEQ ID NO: 4947 attcttaacgggattcctt 5955 6974 1 3 SEQ ID NO: 3607 cattcagaaccagggaaga 3233 3252 SEQ ID NO: 4948 taaagccattcaggtctct 4091 4110 1 3 SEQ ID NO: 3608 acaaactgtttatagtccc 3234 3253 SEQ ID NO: 4949 gacatgtttgaagggtgga 4090 4109 1 3 SEQ ID NO: 3609 ggatttcaatacatttcac 3288 3307 SEQ ID NO: 4950 tgggctaaacgtttaattg 10851 10670 1 3 SEQ ID NO: 3610 ttgtagaaatgaaagtaaa 3289 3308 SEQ ID NO: 4951 tattgaaatatgatttttt 10850 10869 1 3 SEQ ID NO: 3611 ccaaaatttctctcggctt 3305 3324 SEQ ID NO: 4952 agcttgtttgccagtgggc 10052 10071 1 3 SEQ ID NO: 3612 caaattttctctgctaaaa 3371 3390 SEQ ID NO: 4953 ggacaaggcccagaatctg 4215 4234 1 3 SEQ ID NO: 3613 tctgctggaaacaacaacg 3372 3391 SEQ ID NO: 4954 aaagaaacctatggcctta 4062 4081 1 3 SEQ ID NO: 3614 cagaagctaagcaatgtcc 3390 3409 SEQ ID NO: 4955 attcttaacgggattcctt 9076 9095 1 3 SEQ ID NO: 3615 taagattaaatatttactt 3396 3417 SEQ ID NO: 4956 taaagccattcaggtctct 9226 9245 1 3 SEQ ID NO: 3616 aaagattactttgagaaat 3435 3454 SEQ ID NO: 4957 taaagccattcaggtctct 5835 9654 1 3 SEQ ID NO: 3617 aggaaatagttggattacc 3438 3457 SEQ ID NO: 4958 gacatgtttgaagggtgga 6316 6335 1 3 SEQ ID NO: 3618 attttgatgatgctggccc 3449 3468 SEQ ID NO: 4959 tgggctaaacgtttaattg 13838 13857 1 3 SEQ ID NO: 3619 gaattatcttttaaacatt 3454 3473 SEQ ID NO: 4960 tattgaaatatgatttttt 10220 10239 1 3 SEQ ID NO: 3620 ttaccaccagtttgaagtt 3455 3474 SEQ ID NO: 4961 agcttgtttgccagtgggc 10219 10238 1 3 SEQ ID NO: 3621 taagattaaatatttactt 3463 3482 SEQ ID NO: 4962 ggacaaggcccagaatctg 11348 11967 1 3 SEQ ID NO: 3622 aaagattactttgagaaat 3473 3492 SEQ ID NO: 4963 aaagaaacctatggcctta 7084 7103 1 3 SEQ ID NO: 3623 aggaaatagttggattacc 3491 3510 SEQ ID NO: 4964 attcttaacgggattcctt 8858 5877 1 3 SEQ ID NO: 3624 attttgatgatgctggccc 3504 3523 SEQ ID NO: 4965 aattgttgaaagaaaaact 5185 5204 1 3 SEQ ID NO: 3625 aggtttaaatggataattt 3510 3529 SEQ ID NO: 4966 ggacaaggcccagaatctg 5633 5652 1 3 SEQ ID NO: 3626 cagaagctaagcaatgtcc 3523 3542 SEQ ID NO: 4967 aaagaaacctatggcctta 8341 8360 1 3 SEQ ID NO: 3627 taagattaaatatttactt 3540 3559 SEQ ID NO: 4968 attcttaacgggattcctt 13947 13968 1 3 SEQ ID NO: 3628 aaagattactttgagaaat 3555 3574 SEQ ID NO: 4969 taaagccattcaggtctct 9701 9720 1 3 SEQ ID NO: 3629 aggaaatagttggattacc 3507 3588 SEQ ID NO: 4970 gacatgtttgaagggtgga 8167 8188 1 3 SEQ ID NO: 3630 attttgatgatgctggccc 3578 3597 SEQ ID NO: 4971 tgggctaaacgtttaattg 8211 8230 1 3 SEQ ID NO: 3631 gaattatcttttaaacatt 3610 3629 SEQ ID NO: 4972 tattgaaatatgatttttt 8947 8999 1 3 SEQ ID NO: 3632 ttaccaccagtttgaagtt 3631 3650 SEQ ID NO: 4973 agcttgtttgccagtgggc 7713 7732 1 3 SEQ ID NO: 3633 ttgcagtgtatcggacaga 3657 3676 SEQ ID NO: 4974 tgaaatcattgaaaattta 3736 3755 1 3 SEQ ID NO: 3634 cattcagaaccagggaaga 3685 3704 SEQ ID NO: 4975 caggaggctttaagttgtc 5195 5214 1 3 SEQ ID NO: 3635 acaaactgtttatagtccc 3764 3783 SEQ ID NO: 4976 agcttgtttgccagtgggc 12777 12796 1 3 SEQ ID NO: 3636 ggatttcaatacatttcac 3770 3789 SEQ ID NO: 4977 taaagccattcaggtctct 7022 7041 1 3 SEQ ID NO: 3637 ttgtagaaatgaaagtaaa 3809 3828 SEQ ID NO: 4978 aattgttgaaagaaaaact 6693 6712 1 3 SEQ ID NO: 3638 ctgaacagtgagctgcagt 3829 3848 SEQ ID NO: 4979 taaagccattcaggtctct 12957 12976 1 3 SEQ ID NO: 3639 aatccaatctcctctttgc 3895 3914 SEQ ID NO: 4980 ttaatctttcataagtcaa 6314 6333 1 3 SEQ ID NO: 3640 atttgattttcaagcaaag 3940 3959 SEQ ID NO: 4981 ggacaaggcccagaatctg 12179 12192 1 3 SEQ ID NO: 3641 taagattaaatatttactt 3942 3961 SEQ ID NO: 4982 attcttaacgggattcctt 9213 6232 1 3 SEQ ID NO: 3642 aaagattactttgagaaat 3555 3974 SEQ ID NO: 4983 aattgttgaaagaaaaact 6343 6263 1 3 SEQ ID NO: 3643 aggaaatagttggattacc 3971 3990 SEQ ID NO: 4984 agcttgtttgccagtgggc 7363 7382 1 3 SEQ ID NO: 3644 attttgatgatgctggccc 3984 4003 SEQ ID NO: 4985 ttaatctttcataagtcaa 8310 8329 1 3 SEQ ID NO: 3645 gaattatcttttaaacatt 3995 4014 SEQ ID NO: 4986 taaagccattcaggtctct 6047 9066 1 3 SEQ ID NO: 3646 ttaccaccagtttgaagtt 3996 4015 SEQ ID NO: 4987 aattgttgaaagaaaaact 10454 10473 1 3 SEQ ID NO: 3647 ttgcagtgtatcggacaga 3958 4017 SEQ ID NO: 4988 taaagccattcaggtctct 12030 12049 1 3 SEQ ID NO: 3648 cattcagaaccagggaaga 4046 4065 SEQ ID NO: 4989 attcttaacgggattcctt 10545 10564 1 3 SEQ ID NO: 3649 acaaactgtttatagtccc 4052 4071 SEQ ID NO: 4990 taaagccattcaggtctct 8073 8092 1 3 SEQ ID NO: 3650 ggatttcaatacatttcac 4074 4093 SEQ ID NO: 4991 gacatgtttgaagggtgga 8813 8832 1 3 SEQ ID NO: 3651 ttgtagaaatgaaagtaaa 4075 4094 SEQ ID NO: 4992 tgggctaaacgtttaattg 5728 6747 1 3 SEQ ID NO: 3652 ctgaacagtgagctgcagt 4102 4121 SEQ ID NO: 4993 tattgaaatatgatttttt 5330 5349 1 3 SEQ ID NO: 3653 aatccaatctcctctttgc 4142 4161 SEQ ID NO: 4994 agcttgtttgccagtgggc 5254 6273 1 3 SEQ ID NO: 3654 atttgattttcaagcaaag 4156 4175 SEQ ID NO: 4995 tgaaatcattgaaaattta 8843 8862 1 3 SEQ ID NO: 3655 ttttgattttcaagcaaat 4176 1497 SEQ ID NO: 4996 caggaggctttaagttgtc 4735 4754 1 3 SEQ ID NO: 3656 tgatttcaagcaaatgcag 4192 4211 SEQ ID NO: 4997 agcttgtttgccagtgggc 5588 8807 1 3 SEQ ID NO: 3657 atgctgtttgggaaattgg 4195 4214 SEQ ID NO: 4998 taaagccattcaggtctct 11671 11690 1 3 SEQ ID NO: 3658 ttgtagaaatgaaagtaaa 4196 4215 SEQ ID NO: 4999 aattgttgaaagaaaaact 11670 11880 1 3 SEQ ID NO: 3659 ctgaacagtgagctgcagt 4232 4251 SEQ ID NO: 5000 ttaatctttcataagtcaa 13359 13378 1 3 SEQ ID NO: 3660 ggatttcaatacatttcac 4280 4299 SEQ ID NO: 5001 taaagccattcaggtctct 8611 8630 1 3 SEQ ID NO: 3661 ttgtagaaatgaaagtaaa 4291 4310 SEQ ID NO: 5002 aattgttgaaagaaaaact 13189 13188 1 3 SEQ ID NO: 3662 ctgaacagtgagctgcagt 4303 4322 SEQ ID NO: 5003 taaagccattcaggtctct 6966 6984 1 3 SEQ ID NO: 3663 ctgaaaaagttagtgaaag 4312 4331 SEQ ID NO: 5004 agcttgtttgccagtgggc 11066 11107 1 3 SEQ ID NO: 3664 tttttcccagacagtgtca 4313 4332 SEQ ID NO: 5005 taaagccattcaggtctct 11087 11106 1 3 SEQ ID NO: 3665 ttttcccagacagtgtcca 4319 4338 SEQ ID NO: 5006 aattgttgaaagaaaaact 8577 8596 1 3 SEQ ID NO: 3666 cattcagaaccagggaaga 4352 4371 SEQ ID NO: 5007 taaagccattcaggtctct 9984 10003 1 3 SEQ ID NO: 3667 acaaactgtttatagtccc 4353 4372 SEQ ID NO: 5008 ttaatctttcataagtcaa 9933 10002 1 3 SEQ ID NO: 3668 ggatttcaatacatttcac 4363 4382 SEQ ID NO: 5009 ggacaaggcccagaatctg 6487 8506 1 3 SEQ ID NO: 3669 ttgtagaaatgaaagtaaa 4406 4425 SEQ ID NO: 5010 attcttaacgggattcctt 6685 6704 1 3 SEQ ID NO: 3670 ccaaaatttctctcggctt 4412 4431 SEQ ID NO: 5011 aattgttgaaagaaaaact 7102 7121 1 3 SEQ ID NO: 3671 caaattttctctgctaaaa 4449 4468 SEQ ID NO: 5012 agcttgtttgccagtgggc 9214 9233 1 3 SEQ ID NO: 3672 tctgctggaaacaacaacg 4456 4475 SEQ ID NO: 5013 ttaatctttcataagtcaa 11302 11321 1 3 SEQ ID NO: 3673 ttgtagaaatgaaagtaaa 4462 4481 SEQ ID NO: 5014 taaagccattcaggtctct 12231 12250 1 3 SEQ ID NO: 3674 ccaaaatttctctcggctt 4463 4482 SEQ ID NO: 5015 aattgttgaaagaaaaact 12230 12249 1 3 SEQ ID NO: 3675 caaattttctctgctaaaa 4465 4484 SEQ ID NO: 5016 taaagccattcaggtctct 9280 9299 1 3 SEQ ID NO: 3676 tctgctggaaacaacaacg 4543 4562 SEQ ID NO: 5017 attcttaacgggattcctt 11214 11233 1 3 SEQ ID NO: 3677 cagaagctaagcaatgtcc 4551 4570 SEQ ID NO: 5018 taaagccattcaggtctct 8022 8041 1 3 SEQ ID NO: 3678 taagattaaatatttactt 4559 4588 SEQ ID NO: 5019 gacatgtttgaagggtgga 13422 13441 1 3 SEQ ID NO: 3679 aaagattactttgagaaat 4588 4605 SEQ ID NO: 5020 tgggctaaacgtttaattg 11995 12014 1 3 SEQ ID NO: 3680 aggaaatagttggattacc 4588 4607 SEQ ID NO: 5021 tattgaaatatgatttttt 8873 8882 1 3 SEQ ID NO: 3681 attttgatgatgctggccc 4505 4624 SEQ ID NO: 5022 agcttgtttgccagtgggc 9245 9264 1 3 SEQ ID NO: 3682 gaattatcttttaaacatt 4614 4633 SEQ ID NO: 5023 tgaaatcattgaaaattta 9372 9391 1 3 SEQ ID NO: 3683 ttaccaccagtttgaagtt 4667 4686 SEQ ID NO: 5024 caggaggctttaagttgtc 6190 6209 1 3 SEQ ID NO: 3684 taagattaaatatttactt 4668 4587 SEQ ID NO: 5025 agcttgtttgccagtgggc 9308 9327 1 3 SEQ ID NO: 3685 aaagattactttgagaaat 4892 4711 SEQ ID NO: 5026 taaagccattcaggtctct 9376 9385 1 3 SEQ ID NO: 3686 aggaaatagttggattacc 4730 4749 SEQ ID NO: 5027 aattgttgaaagaaaaact 12291 12310 1 3 SEQ ID NO: 3687 attttgatgatgctggccc 4782 4781 SEQ ID NO: 5028 taaagccattcaggtctct 12263 12282 1 3 SEQ ID NO: 3688 aggtttaaatggataattt 4783 4782 SEQ ID NO: 5029 ttaatctttcataagtcaa 12262 12281 1 3 SEQ ID NO: 3689 cagaagctaagcaatgtcc 4793 4812 SEQ ID NO: 5030 ggacaaggcccagaatctg 5977 5996 1 3 SEQ ID NO: 3690 taagattaaatatttactt 4804 4823 SEQ ID NO: 5031 attcttaacgggattcctt 8620 6939 1 3 SEQ ID NO: 3691 aaagattactttgagaaat 4858 4887 SEQ ID NO: 5032 aattgttgaaagaaaaact 13032 13051 1 3 SEQ ID NO: 3692 aggaaatagttggattacc 4907 4926 SEQ ID NO: 5033 agcttgtttgccagtgggc 6901 6920 1 3 SEQ ID NO: 3693 attttgatgatgctggccc 4940 4959 SEQ ID NO: 5034 ttaatctttcataagtcaa 5348 5367 1 3 SEQ ID NO: 3694 gaattatcttttaaacatt 4963 4982 SEQ ID NO: 5035 taaagccattcaggtctct 12549 12568 1 3 SEQ ID NO: 3695 ttaccaccagtttgaagtt 4981 5000 SEQ ID NO: 5036 aattgttgaaagaaaaact 10103 10122 1 3 SEQ ID NO: 3696 ttgcagtgtatcggacaga 5007 5026 SEQ ID NO: 5037 taaagccattcaggtctct 14019 14038 1 3 SEQ ID NO: 3697 cattcagaaccagggaaga 5040 5059 SEQ ID NO: 5038 agcttgtttgccagtgggc 6794 6813 1 3 SEQ ID NO: 3698 acaaactgtttatagtccc 5083 5102 SEQ ID NO: 5039 ggacaaggcccagaatctg 5185 5204 1 3 SEQ ID NO: 3699 ggatttcaatacatttcac 5092 5111 SEQ ID NO: 5040 aaagaaacctatggcctta 2239 6258 1 3 SEQ ID NO: 3700 ttgtagaaatgaaagtaaa 5115 5135 SEQ ID NO: 5041 attcttaacgggattcctt 9222 9241 1 3 SEQ ID NO: 3701 ctgaacagtgagctgcagt 5135 5154 SEQ ID NO: 5042 taaagccattcaggtctct 6899 6918 1 3 SEQ ID NO: 3702 aatccaatctcctctttgc 5149 5167 SEQ ID NO: 5043 tattgaaatatgatttttt 6499 6518 1 3 SEQ ID NO: 3703 atttgattttcaagcaaag 5150 5189 SEQ ID NO: 5044 agcttgtttgccagtgggc 6436 6455 1 3 SEQ ID NO: 3704 ttttgattttcaagcaaat 5193 5212 SEQ ID NO: 5045 ggacaaggcccagaatctg 7506 7525 1 3 SEQ ID NO: 3705 tgatttcaagcaaatgcag 5231 5250 SEQ ID NO: 5046 ggacaaggcccagaatctg 8149 8168 1 3 SEQ ID NO: 3706 atgctgtttgggaaattgg 5247 5266 SEQ ID NO: 5047 attcttaacgggattcctt 11358 11375 1 3 SEQ ID NO: 3707 tgctgttttttggaaatgc 5303 5322 SEQ ID NO: 5048 aattgttgaaagaaaaact 11977 11966 1 3 SEQ ID NO: 3708 aaaaaaatacactggacgt 5336 5355 SEQ ID NO: 5049 agcttgtttgccagtgggc 6486 6605 1 3 SEQ ID NO: 3709 actggagcttagtaagggc 5349 5366 SEQ ID NO: 5050 ttaatctttcataagtcaa 5746 5765 1 3 SEQ ID NO: 3710 cttctggaaagagggtcat 5353 5372 SEQ ID NO: 5051 taaagccattcaggtctct 6702 6721 1 3 SEQ ID NO: 3711 ggaaaagggtcatggaaat 5386 5405 SEQ ID NO: 5052 aattgttgaaagaaaaact 10996 11015 1 3 SEQ ID NO: 3712 gggcctgccccagatttct 5389 5408 SEQ ID NO: 5053 taaagccattcaggtctct 12490 12509 1 3 SEQ ID NO: 3713 ttctcagataggggaacac 5395 5414 SEQ ID NO: 5054 ttaatctttcataagtcaa 11852 11871 1 3 SEQ ID NO: 3714 gatgaaagaattaagggga 5420 5439 SEQ ID NO: 5055 ggacaaggcccagaatctg 7370 7389 1 3 SEQ ID NO: 3715 cttggactgtcaaataagt 5435 5484 SEQ ID NO: 5056 attcttaacgggattcctt 8022 8041 1 3 SEQ ID NO: 3716 ggaaaagggtcatggaaat 5443 5482 SEQ ID NO: 5057 aattgttgaaagaaaaact 10674 10693 1 3 SEQ ID NO: 3717 aggaaatagttggattacc 5468 5487 SEQ ID NO: 5058 agcttgtttgccagtgggc 5578 5597 1 3 SEQ ID NO: 3718 attttgatgatgctggccc 5491 5510 SEQ ID NO: 5059 ttaatctttcataagtcaa 7275 7294 1 3 SEQ ID NO: 3719 gaattatcttttaaacatt 5596 5615 SEQ ID NO: 5060 agcttgtttgccagtgggc 9375 9395 1 3 SEQ ID NO: 3720 ttaccaccagtttgaagtt 5599 5618 SEQ ID NO: 5061 tgaaatcattgaaaattta 6271 6290 1 3 SEQ ID NO: 3721 ctgaaaaagttagtgaaag 5602 5621 SEQ ID NO: 5062 caggaggctttaagttgtc 10127 10148 1 3 SEQ ID NO: 3722 tttttcccagacagtgtca 6816 5635 SEQ ID NO: 5063 agcttgtttgccagtgggc 7065 7084 1 3 SEQ ID NO: 3723 ttttcccagacagtgtcca 5620 5845 SEQ ID NO: 5064 taaagccattcaggtctct 12158 12177 1 3 SEQ ID NO: 3724 cattcagaaccagggaaga 6586 5705 SEQ ID NO: 5065 cttcatcgttcatttgagt 9087 9106 1 3 SEQ ID NO: 3725 ttgcagtgtatcggacaga 5705 5724 SEQ ID NO: 5066 cttatgggatttcctaagg 7450 7489 1 3 SEQ ID NO: 3726 cattcagaaccagggaaga 5740 5759 SEQ ID NO: 5067 cttcatcgttcatttgagt 8598 9816 1 3 SEQ ID NO: 3727 acaaactgtttatagtccc 5790 5809 SEQ ID NO: 5068 ttccatcacaaatcctttg 8828 8647 1 3 SEQ ID NO: 3728 ggatttcaatacatttcac 5791 5810 SEQ ID NO: 5069 tctcaaagagttacaacag 8627 8646 1 3 SEQ ID NO: 3729 ttgtagaaatgaaagtaaa 5792 5811 SEQ ID NO: 5070 tattgaaatatgatttttt 8626 8645 1 3 SEQ ID NO: 3730 ctgaacagtgagctgcagt 5794 5813 SEQ ID NO: 5071 aattgttgaaagaaaaact 12412 12431 1 3 SEQ ID NO: 3731 aatccaatctcctctttgc 5800 5819 SEQ ID NO: 5072 ggacaaggcccagaatctg 6501 8520 1 3 SEQ ID NO: 3732 atttgattttcaagcaaag 5801 5820 SEQ ID NO: 5073 aaagaaacctatggcctta 12975 12994 1 3 SEQ ID NO: 3733 ttttgattttcaagcaaat 5859 5878 SEQ ID NO: 5074 attcttaacgggattcctt 12213 12232 1 3 SEQ ID NO: 3734 tgatttcaagcaaatgcag 5879 5898 SEQ ID NO: 5075 taaagccattcaggtctct 1088 1107 1 3 SEQ ID NO: 3735 atgctgtttgggaaattgg 5914 5933 SEQ ID NO: 5076 gacatgtttgaagggtgga 8274 8293 1 3 SEQ ID NO: 3736 tgctgttttttggaaatgc 5983 6002 SEQ ID NO: 5077 tgggctaaacgtttaattg 13612 13631 1 3 SEQ ID NO: 3737 aaaaaaatacactggacgt 5993 6012 SEQ ID NO: 5078 tattgaaatatgatttttt 13351 13370 1 3 SEQ ID NO: 3738 ctgaaaaagttagtgaaag 6009 6028 SEQ ID NO: 5079 agcttgtttgccagtgggc 6418 6437 1 3 SEQ ID NO: 3739 tttttcccagacagtgtca 6046 6085 SEQ ID NO: 5080 aaagaaacctatggcctta 11610 11629 1 3 SEQ ID NO: 3740 ttttcccagacagtgtcca 6061 6080 SEQ ID NO: 5081 attcttaacgggattcctt 8037 8058 1 3 SEQ ID NO: 3741 cattcagaaccagggaaga 6084 6103 SEQ ID NO: 5082 taaagccattcaggtctct 9882 9701 1 3 SEQ ID NO: 3742 acaaactgtttatagtccc 6103 6122 SEQ ID NO: 5083 gacatgtttgaagggtgga 8599 8618 1 3 SEQ ID NO: 3743 ggatttcaatacatttcac 6132 6151 SEQ ID NO: 5084 tgggctaaacgtttaattg 8273 8292 1 3 SEQ ID NO: 3744 ttgtagaaatgaaagtaaa 6198 6217 SEQ ID NO: 5085 tattgaaatatgatttttt 8988 7007 1 3 SEQ ID NO: 3745 ccaaaatttctctcggctt 6235 6254 SEQ ID NO: 5086 agcttgtttgccagtgggc 9177 9196 1 3 SEQ ID NO: 3746 caaattttctctgctaaaa 6257 6276 SEQ ID NO: 5087 ggacaaggcccagaatctg 12970 12989 1 3 SEQ ID NO: 3747 tctgctggaaacaacaacg 6276 6295 SEQ ID NO: 5088 aaagaaacctatggcctta 10187 10208 1 3 SEQ ID NO: 3748 cagaagctaagcaatgtcc 6266 6305 SEQ ID NO: 5089 attcttaacgggattcctt 13655 13374 1 3 SEQ ID NO: 3749 taagattaaatatttactt 6288 6307 SEQ ID NO: 5090 taaagccattcaggtctct 6358 6377 1 3 SEQ ID NO: 3750 aaagattactttgagaaat 6315 6334 SEQ ID NO: 5091 taaagccattcaggtctct 9576 9595 1 3 SEQ ID NO: 3751 aggaaatagttggattacc 6337 6358 SEQ ID NO: 5092 gacatgtttgaagggtgga 9556 9586 1 3 SEQ ID NO: 3752 attttgatgatgctggccc 6344 6353 SEQ ID NO: 5093 tgggctaaacgtttaattg 12948 12967 1 3 SEQ ID NO: 3753 gaattatcttttaaacatt 6423 6442 SEQ ID NO: 5094 tattgaaatatgatttttt 11829 11848 1 3 SEQ ID NO: 3754 ttaccaccagtttgaagtt 6451 6470 SEQ ID NO: 5095 agcttgtttgccagtgggc 13622 13641 1 3 SEQ ID NO: 3755 taagattaaatatttactt 3460 6479 SEQ ID NO: 5096 ggacaaggcccagaatctg 7724 7743 1 3 SEQ ID NO: 3756 aaagattactttgagaaat 6489 6480 SEQ ID NO: 5097 aaagaaacctatggcctta 12869 12888 1 3 SEQ ID NO: 3757 aggaaatagttggattacc 8497 6516 SEQ ID NO: 5098 attcttaacgggattcctt 10479 10498 1 3 SEQ ID NO: 3758 attttgatgatgctggccc 6503 6522 SEQ ID NO: 5099 aattgttgaaagaaaaact 11809 11827 1 3 SEQ ID NO: 3759 aggtttaaatggataattt 6534 6553 SEQ ID NO: 5100 ggacaaggcccagaatctg 11163 11182 1 3 SEQ ID NO: 3760 cagaagctaagcaatgtcc 6541 6680 SEQ ID NO: 5101 aaagaaacctatggcctta 8380 8399 1 3 SEQ ID NO: 3761 taagattaaatatttactt 6544 6563 SEQ ID NO: 5102 attcttaacgggattcctt 9695 9714 1 3 SEQ ID NO: 3762 aaagattactttgagaaat 6641 6650 SEQ ID NO: 5103 taaagccattcaggtctct 1933 1962 1 3 SEQ ID NO: 3763 aggaaatagttggattacc 6857 6678 SEQ ID NO: 5104 gacatgtttgaagggtgga 13815 13834 1 3 SEQ ID NO: 3764 attttgatgatgctggccc 6883 6702 SEQ ID NO: 5105 tgggctaaacgtttaattg 13128 13147 1 3 SEQ ID NO: 3765 gaattatcttttaaacatt 6697 6716 SEQ ID NO: 5106 tattgaaatatgatttttt 13854 13683 1 3 SEQ ID NO: 3766 ttaccaccagtttgaagtt 6899 6717 SEQ ID NO: 5107 agcttgtttgccagtgggc 13883 13882 1 3 SEQ ID NO: 3767 ttgcagtgtatcggacaga 6703 6722 SEQ ID NO: 5108 tgaaatcattgaaaattta 14084 14103 1 3 SEQ ID NO: 3768 cattcagaaccagggaaga 6719 6738 SEQ ID NO: 5109 caggaggctttaagttgtc 8268 8287 1 3 SEQ ID NO: 3769 acaaactgtttatagtccc 6747 6766 SEQ ID NO: 5110 agcttgtttgccagtgggc 12864 12883 1 3 SEQ ID NO: 3770 ggatttcaatacatttcac 6748 6767 SEQ ID NO: 5111 taaagccattcaggtctct 8789 8788 1 3 SEQ ID NO: 3771 ttgtagaaatgaaagtaaa 6753 6772 SEQ ID NO: 5112 aattgttgaaagaaaaact 13101 13120 1 3 SEQ ID NO: 3772 ctgaacagtgagctgcagt 6777 6796 SEQ ID NO: 5113 taaagccattcaggtctct 11702 11721 1 3 SEQ ID NO: 3773 aatccaatctcctctttgc 6760 6799 SEQ ID NO: 5114 ttaatctttcataagtcaa 11810 11828 1 3 SEQ ID NO: 3774 atttgattttcaagcaaag 6805 6824 SEQ ID NO: 5115 ggacaaggcccagaatctg 11175 11194 1 3 SEQ ID NO: 3775 ttttgattttcaagcaaat 6524 6843 SEQ ID NO: 5116 attcttaacgggattcctt 9671 9890 1 3 SEQ ID NO: 3776 tgatttcaagcaaatgcag 6828 6847 SEQ ID NO: 5117 aattgttgaaagaaaaact 8014 8033 1 3 SEQ ID NO: 3777 atgctgtttgggaaattgg 6888 6907 SEQ ID NO: 5118 agcttgtttgccagtgggc 10088 10107 1 3 SEQ ID NO: 3778 ttgtagaaatgaaagtaaa 6894 6913 SEQ ID NO: 5119 ttaatctttcataagtcaa 9065 9084 1 3 SEQ ID NO: 3779 attttgatgatgctggccc 6924 6943 SEQ ID NO: 5120 taaagccattcaggtctct 9491 9510 1 3 SEQ ID NO: 3780 gaattatcttttaaacatt 6950 6969 SEQ ID NO: 5121 aattgttgaaagaaaaact 3688 3767 1 3 SEQ ID NO: 3781 ttaccaccagtttgaagtt 7080 7079 SEQ ID NO: 5122 taaagccattcaggtctct 6191 6210 1 3 SEQ ID NO: 3782 ttgcagtgtatcggacaga 7081 7080 SEQ ID NO: 5123 attcttaacgggattcctt 5334 5353 1 3 SEQ ID NO: 3783 cattcagaaccagggaaga 7070 7089 SEQ ID NO: 5124 taaagccattcaggtctct 13133 13152 1 3 SEQ ID NO: 3784 acaaactgtttatagtccc 7111 7130 SEQ ID NO: 5125 gacatgtttgaagggtgga 12029 12048 1 3 SEQ ID NO: 3785 ggatttcaatacatttcac 7160 7179 SEQ ID NO: 5126 tgggctaaacgtttaattg 10331 10350 1 3 SEQ ID NO: 3786 ttgtagaaatgaaagtaaa 7164 7183 SEQ ID NO: 5127 tattgaaatatgatttttt 12158 12175 1 3 SEQ ID NO: 3787 ctgaacagtgagctgcagt 7223 7242 SEQ ID NO: 5128 agcttgtttgccagtgggc 10168 10187 1 3 SEQ ID NO: 3788 aatccaatctcctctttgc 7264 7283 SEQ ID NO: 5129 tgaaatcattgaaaattta 11334 11353 1 3 SEQ ID NO: 3789 atttgattttcaagcaaag 7271 7290 SEQ ID NO: 5130 caggaggctttaagttgtc 2077 2096 1 3 SEQ ID NO: 3790 ttttgattttcaagcaaat 7280 7299 SEQ ID NO: 5131 agcttgtttgccagtgggc 9069 9088 1 3 SEQ ID NO: 3791 tgatttcaagcaaatgcag 7288 7307 SEQ ID NO: 5132 taaagccattcaggtctct 7443 7462 1 3 SEQ ID NO: 3792 atgctgtttgggaaattgg 7292 7311 SEQ ID NO: 5133 aattgttgaaagaaaaact 10419 10438 1 3 SEQ ID NO: 3793 tgctgttttttggaaatgc 7300 7319 SEQ ID NO: 5134 ttaatctttcataagtcaa 9953 9972 1 3 SEQ ID NO: 3794 aaaaaaatacactggacgt 7353 7372 SEQ ID NO: 5135 taaagccattcaggtctct 7422 7441 1 3 SEQ ID NO: 3795 actggagcttagtaagggc 7354 7373 SEQ ID NO: 5136 aattgttgaaagaaaaact 7421 7440 1 3 SEQ ID NO: 3796 cttctggaaagagggtcat 7355 7374 SEQ ID NO: 5137 ttaatctttcataagtcaa 10495 10514 1 3 SEQ ID NO: 3797 ggaaaagggtcatggaaat 7356 7375 SEQ ID NO: 5138 ggacaaggcccagaatctg 10494 10513 1 3 SEQ ID NO: 3798 gggcctgccccagatttct 7357 7376 SEQ ID NO: 5139 attcttaacgggattcctt 10493 10512 1 3 SEQ ID NO: 3799 ttctcagataggggaacac 7380 7399 SEQ ID NO: 5140 aattgttgaaagaaaaact 11487 11606 1 3 SEQ ID NO: 3800 gatgaaagaattaagggga 7406 7425 SEQ ID NO: 5141 agcttgtttgccagtgggc 8791 8810 1 3 SEQ ID NO: 3801 cttggactgtcaaataagt 7441 7460 SEQ ID NO: 5142 ttaatctttcataagtcaa 7972 7991 1 3 SEQ ID NO: 3802 ggaaaagggtcatggaaat 7442 7461 SEQ ID NO: 5143 agcttgtttgccagtgggc 8536 8687 1 3 SEQ ID NO: 3803 aggaaatagttggattacc 7452 7471 SEQ ID NO: 5144 tgaaatcattgaaaattta 10731 10760 1 3 SEQ ID NO: 3804 attttgatgatgctggccc 7473 7492 SEQ ID NO: 5145 caggaggctttaagttgtc 8409 8428 1 3 SEQ ID NO: 3805 gaattatcttttaaacatt 7547 7566 SEQ ID NO: 5146 agcttgtttgccagtgggc 8540 8559 1 3 SEQ ID NO: 3806 aatccaatctcctctttgc 7616 7635 SEQ ID NO: 5147 taaagccattcaggtctct 10173 10192 1 3 SEQ ID NO: 3807 atttgattttcaagcaaag 7641 7660 SEQ ID NO: 5148 cttcatcgttcatttgagt 10213 10232 1 3 SEQ ID NO: 3808 ttttgattttcaagcaaat 7684 7703 SEQ ID NO: 5149 cttatgggatttcctaagg 11021 11040 1 3 SEQ ID NO: 3809 tgatttcaagcaaatgcag 7700 7719 SEQ ID NO: 5150 cttcatcgttcatttgagt 6254 8273 1 3 SEQ ID NO: 3810 atgctgtttgggaaattgg 7722 7741 SEQ ID NO: 5151 ttccatcacaaatcctttg 9377 9396 1 3 SEQ ID NO: 3811 tgctgttttttggaaatgc 7723 7742 SEQ ID NO: 5152 tctcaaagagttacaacag 8810 8829 1 3 SEQ ID NO: 3812 aaaaaaatacactggacgt 7732 7761 SEQ ID NO: 5153 tattgaaatatgatttttt 10535 10554 1 3 SEQ ID NO: 3813 actggagcttagtaagggc 7738 7757 SEQ ID NO: 5154 aattgttgaaagaaaaact 11025 11044 1 3 SEQ ID NO: 3814 cttctggaaagagggtcat 7742 7781 SEQ ID NO: 5155 ggacaaggcccagaatctg 13681 13700 1 3 SEQ ID NO: 3815 ggaaaagggtcatggaaat 7755 7772 SEQ ID NO: 5156 aaagaaacctatggcctta 10283 10302 1 3 SEQ ID NO: 3816 gggcctgccccagatttct 7770 7789 SEQ ID NO: 5157 attcttaacgggattcctt 14026 14045 1 3 SEQ ID NO: 3817 ttctcagataggggaacac 7847 7866 SEQ ID NO: 5158 taaagccattcaggtctct 10208 10227 1 3 SEQ ID NO: 3818 gatgaaagaattaagggga 7846 7857 SEQ ID NO: 5159 gacatgtttgaagggtgga 10207 10226 1 3 SEQ ID NO: 3819 cttggactgtcaaataagt 7858 7867 SEQ ID NO: 5160 tgggctaaacgtttaattg 8959 8978 1 3 SEQ ID NO: 3820 ggaaaagggtcatggaaat 7887 7906 SEQ ID NO: 5161 tattgaaatatgatttttt 7973 7992 1 3 SEQ ID NO: 3821 ttttgattttcaagcaaat 7897 7916 SEQ ID NO: 5162 agcttgtttgccagtgggc 13978 13997 1 3 SEQ ID NO: 3822 tgatttcaagcaaatgcag 7929 7948 SEQ ID NO: 5163 aaagaaacctatggcctta 9588 9507 1 3 SEQ ID NO: 3823 atgctgtttgggaaattgg 8004 8023 SEQ ID NO: 5164 attcttaacgggattcctt 13183 13202 1 3 SEQ ID NO: 3824 tgctgttttttggaaatgc 8913 8032 SEQ ID NO: 5165 taaagccattcaggtctct 6829 6848 1 3 SEQ ID NO: 3825 aaaaaaatacactggacgt 8039 8058 SEQ ID NO: 5166 gacatgtttgaagggtgga 9472 9491 1 3 SEQ ID NO: 3826 actggagcttagtaagggc 8053 8082 SEQ ID NO: 5167 tgggctaaacgtttaattg 9887 9908 1 3 SEQ ID NO: 3827 cttctggaaagagggtcat 8089 8108 SEQ ID NO: 5168 tattgaaatatgatttttt 10414 10433 1 3 SEQ ID NO: 3828 ggaaaagggtcatggaaat 8145 8164 SEQ ID NO: 5169 agcttgtttgccagtgggc 12932 12861 1 3 SEQ ID NO: 3829 gggcctgccccagatttct 8233 8232 SEQ ID NO: 5170 ggacaaggcccagaatctg 8522 8541 1 3 SEQ ID NO: 3830 ttctcagataggggaacac 8320 8339 SEQ ID NO: 5171 aaagaaacctatggcctta 8884 8903 1 3 SEQ ID NO: 3831 gatgaaagaattaagggga 8339 8358 SEQ ID NO: 5172 attcttaacgggattcctt 8921 8940 1 3 SEQ ID NO: 3832 cttggactgtcaaataagt 8387 8408 SEQ ID NO: 5173 taaagccattcaggtctct 11612 11631 1 3 SEQ ID NO: 3833 ggaaaagggtcatggaaat 8300 8418 SEQ ID NO: 5174 taaagccattcaggtctct 11552 11571 1 3 SEQ ID NO: 3834 aggaaatagttggattacc 8422 8441 SEQ ID NO: 5175 gacatgtttgaagggtgga 9738 9757 1 3 SEQ ID NO: 3835 attttgatgatgctggccc 8436 8459 SEQ ID NO: 5176 tgggctaaacgtttaattg 9670 9889 1 3 SEQ ID NO: 3836 gaattatcttttaaacatt 8508 8527 SEQ ID NO: 5177 tattgaaatatgatttttt 13410 13429 1 3 SEQ ID NO: 3837 ttaccaccagtttgaagtt 8509 8528 SEQ ID NO: 5178 agcttgtttgccagtgggc 13409 13428 1 3 SEQ ID NO: 3838 ctgaaaaagttagtgaaag 8527 8546 SEQ ID NO: 5179 ggacaaggcccagaatctg 11537 11556 1 3 SEQ ID NO: 3839 tttttcccagacagtgtca 8530 8549 SEQ ID NO: 5180 aaagaaacctatggcctta 13650 13679 1 3 SEQ ID NO: 3840 ttttcccagacagtgtcca 8549 8655 SEQ ID NO: 5181 attcttaacgggattcctt 12460 12479 1 3 SEQ ID NO: 3841 cattcagaaccagggaaga 8604 8623 SEQ ID NO: 5182 aattgttgaaagaaaaact 113688 11407 1 3 SEQ ID NO: 3842 ttgcagtgtatcggacaga 8615 8635 SEQ ID NO: 5183 ggacaaggcccagaatctg 13780 13799 1 3 SEQ ID NO: 3843 cattcagaaccagggaaga 8678 8697 SEQ ID NO: 5184 aaagaaacctatggcctta 13422 13441 1 3 SEQ ID NO: 3844 acaaactgtttatagtccc 8751 8770 SEQ ID NO: 5185 attcttaacgggattcctt 13380 13399 1 3 SEQ ID NO: 3845 ggatttcaatacatttcac 8765 8784 SEQ ID NO: 5186 taaagccattcaggtctct 12702 12721 1 3 SEQ ID NO: 3846 ttgtagaaatgaaagtaaa 8829 8848 SEQ ID NO: 5187 gacatgtttgaagggtgga 12259 12278 1 3 SEQ ID NO: 3847 ctgaacagtgagctgcagt 8929 8948 SEQ ID NO: 5188 tgggctaaacgtttaattg 10036 10057 1 3 SEQ ID NO: 3848 aatccaatctcctctttgc 9060 9079 SEQ ID NO: 5189 tattgaaatatgatttttt 12618 12837 1 3 SEQ ID NO: 3849 atttgattttcaagcaaag 9067 9066 SEQ ID NO: 5190 agcttgtttgccagtgggc 7308 7327 1 3 SEQ ID NO: 3850 ttttgattttcaagcaaat 9077 9998 SEQ ID NO: 5191 tgaaatcattgaaaattta 13585 13585 1 3 SEQ ID NO: 3851 tgatttcaagcaaatgcag 9141 9160 SEQ ID NO: 5192 caggaggctttaagttgtc 12506 12525 1 3 SEQ ID NO: 3852 atgctgtttgggaaattgg 9142 9161 SEQ ID NO: 5193 agcttgtttgccagtgggc 2098 2917 1 3 SEQ ID NO: 3853 tgctgttttttggaaatgc 9221 9240 SEQ ID NO: 5194 taaagccattcaggtctct 13638 13557 1 3 SEQ ID NO: 3854 aaaaaaatacactggacgt 9230 6249 SEQ ID NO: 5195 aattgttgaaagaaaaact 13404 13513 1 3 SEQ ID NO: 3855 ctgaaaaagttagtgaaag 9283 9602 SEQ ID NO: 5196 taaagccattcaggtctct 10380 10399 1 3 SEQ ID NO: 3856 tttttcccagacagtgtca 9312 9331 SEQ ID NO: 5197 ttaatctttcataagtcaa 9848 9667 1 3 SEQ ID NO: 3857 ttttcccagacagtgtcca 9405 9424 SEQ ID NO: 5198 ggacaaggcccagaatctg 14038 14057 1 3 SEQ ID NO: 3858 cattcagaaccagggaaga 9435 9454 SEQ ID NO: 5199 attcttaacgggattcctt 13380 13399 1 3 SEQ ID NO: 3859 acaaactgtttatagtccc 9463 9482 SEQ ID NO: 5200 aattgttgaaagaaaaact 11734 11753 1 3 SEQ ID NO: 3860 ggatttcaatacatttcac 9476 9497 SEQ ID NO: 5201 agcttgtttgccagtgggc 11719 11738 1 3 SEQ ID NO: 3861 ttgtagaaatgaaagtaaa 9502 9521 SEQ ID NO: 5202 ttaatctttcataagtcaa 13206 13225 1 3 SEQ ID NO: 3862 ccaaaatttctctcggctt 9505 9524 SEQ ID NO: 5203 taaagccattcaggtctct 7621 9540 1 3 SEQ ID NO: 3863 caaattttctctgctaaaa 9534 9553 SEQ ID NO: 5204 aattgttgaaagaaaaact 11211 11230 1 3 SEQ ID NO: 3864 tctgctggaaacaacaacg 9561 9580 SEQ ID NO: 5205 taaagccattcaggtctct 3936 3955 1 3 SEQ ID NO: 3865 cagaagctaagcaatgtcc 9578 9597 SEQ ID NO: 5206 attcttaacgggattcctt 13021 13040 1 3 SEQ ID NO: 3866 taagattaaatatttactt 9590 9809 SEQ ID NO: 5207 taaagccattcaggtctct 10422 10441 1 3 SEQ ID NO: 3867 aaagattactttgagaaat 9591 9610 SEQ ID NO: 5208 gacatgtttgaagggtgga 11291 11310 1 3 SEQ ID NO: 3868 aggaaatagttggattacc 9640 9659 SEQ ID NO: 5209 tgggctaaacgtttaattg 9805 9824 1 3 SEQ ID NO: 3869 attttgatgatgctggccc 9720 9739 SEQ ID NO: 5210 tattgaaatatgatttttt 11688 11707 1 3 SEQ ID NO: 3870 gaattatcttttaaacatt 9769 9608 SEQ ID NO: 5211 agcttgtttgccagtgggc 12762 12781 1 3 SEQ ID NO: 3871 ttaccaccagtttgaagtt 9859 9878 SEQ ID NO: 5212 tgaaatcattgaaaattta 8938 8957 1 3 SEQ ID NO: 3872 taagattaaatatttactt 9876 9595 SEQ ID NO: 5213 caggaggctttaagttgtc 13207 13226 1 3 SEQ ID NO: 3873 aaagattactttgagaaat 9594 9913 SEQ ID NO: 5214 agcttgtttgccagtgggc 4842 4861 1 3 SEQ ID NO: 3874 aggaaatagttggattacc 9950 9969 SEQ ID NO: 5215 taaagccattcaggtctct 11080 11099 1 3 SEQ ID NO: 3875 attttgatgatgctggccc 10113 10132 SEQ ID NO: 5216 aattgttgaaagaaaaact 5440 8469 1 3 SEQ ID NO: 3876 aggtttaaatggataattt 10167 10186 SEQ ID NO: 5217 ttaatctttcataagtcaa 7224 7243 1 3 SEQ ID NO: 3877 cagaagctaagcaatgtcc 10201 10220 SEQ ID NO: 5218 tattgaaatatgatttttt 12889 12906 1 3 SEQ ID NO: 3878 taagattaaatatttactt 10204 10223 SEQ ID NO: 5219 agcttgtttgccagtgggc 12431 12450 1 3 SEQ ID NO: 3879 aaagattactttgagaaat 10232 10251 SEQ ID NO: 5220 tgaaatcattgaaaattta 13928 13947 1 3 SEQ ID NO: 3880 aggaaatagttggattacc 10305 10324 SEQ ID NO: 5221 caggaggctttaagttgtc 8195 8214 1 3 SEQ ID NO: 3881 attttgatgatgctggccc 10330 10349 SEQ ID NO: 5222 agcttgtttgccagtgggc 7161 7180 1 3 SEQ ID NO: 3882 gaattatcttttaaacatt 10377 10306 SEQ ID NO: 5223 taaagccattcaggtctct 10778 10797 1 3 SEQ ID NO: 3883 ttaccaccagtttgaagtt 10453 10472 SEQ ID NO: 5224 aattgttgaaagaaaaact 13023 13042 1 3 SEQ ID NO: 3884 ttgcagtgtatcggacaga 10463 10452 SEQ ID NO: 5225 taaagccattcaggtctct 12859 12878 1 3 SEQ ID NO: 3885 cattcagaaccagggaaga 10476 10494 SEQ ID NO: 5226 ttaatctttcataagtcaa 10824 10943 1 3 SEQ ID NO: 3886 acaaactgtttatagtccc 10482 10501 SEQ ID NO: 5227 ggacaaggcccagaatctg 7148 7167 1 3 SEQ ID NO: 3887 ggatttcaatacatttcac 10512 10531 SEQ ID NO: 5228 attcttaacgggattcctt 12242 1226+1 1 3 SEQ ID NO: 3888 ttgtagaaatgaaagtaaa 10648 10567 SEQ ID NO: 5231 aattgttgaaagaaaaact 12036 12055 1 3 SEQ ID NO: 3889 ctgaacagtgagctgcagt 10573 10592 SEQ ID NO: 5232 agcttgtttgccagtgggc 10649 10668 1 3 SEQ ID NO: 3890 aatccaatctcctctttgc 10710 10729 SEQ ID NO: 5233 ttaatctttcataagtcaa 12289 12308 1 3 SEQ ID NO: 3891 atttgattttcaagcaaag 10723 10742 SEQ ID NO: 5234 taaagccattcaggtctct 12745 12764 1 3 SEQ ID NO: 3892 ttttgattttcaagcaaat 10880 10879 SEQ ID NO: 5235 aattgttgaaagaaaaact 2467 2486 1 3 SEQ ID NO: 3893 tgatttcaagcaaatgcag 10897 10916 SEQ ID NO: 5236 taaagccattcaggtctct 12950 12979 1 3 SEQ ID NO: 3894 atgctgtttgggaaattgg 10898 10917 SEQ ID NO: 5237 attcttaacgggattcctt 12959 12978 1 3 SEQ ID NO: 3895 ttgtagaaatgaaagtaaa 10914 10933 SEQ ID NO: 5238 taaagccattcaggtctct 14066 14085 1 3 SEQ ID NO: 3896 attttgatgatgctggccc 10915 10934 SEQ ID NO: 5239 gacatgtttgaagggtgga 14085 14084 1 3 SEQ ID NO: 3897 gaattatcttttaaacatt 10940 10959 SEQ ID NO: 5240 tgggctaaacgtttaattg 12258 12276 1 3 SEQ ID NO: 3898 ttaccaccagtttgaagtt 10987 11006 SEQ ID NO: 5241 tattgaaatatgatttttt 11186 11205 1 3 SEQ ID NO: 3899 ttgcagtgtatcggacaga 10994 11013 SEQ ID NO: 5242 agcttgtttgccagtgggc 13347 13368 1 3 SEQ ID NO: 3900 cattcagaaccagggaaga 10995 11014 SEQ ID NO: 5243 tgaaatcattgaaaattta 13346 13366 1 3 SEQ ID NO: 3901 acaaactgtttatagtccc 11003 11022 SEQ ID NO: 5244 caggaggctttaagttgtc 13622 13641 1 3 SEQ ID NO: 3902 ggatttcaatacatttcac 11016 11037 SEQ ID NO: 5245 agcttgtttgccagtgggc 9820 9299 1 3 SEQ ID NO: 3903 aaagattactttgagaaat 11032 11051 SEQ ID NO: 5246 taaagccattcaggtctct 13166 13185 1 3 SEQ ID NO: 3904 aggaaatagttggattacc 11079 11098 SEQ ID NO: 5247 aattgttgaaagaaaaact 10466 10485 1 3 SEQ ID NO: 3905 attttgatgatgctggccc 11115 11134 SEQ ID NO: 5248 ttaatctttcataagtcaa 7572 7591 1 3 SEQ ID NO: 3906 aggtttaaatggataattt 11199 11216 SEQ ID NO: 5249 taaagccattcaggtctct 12370 12389 1 3 SEQ ID NO: 3907 cagaagctaagcaatgtcc 11239 11258 SEQ ID NO: 5250 aattgttgaaagaaaaact 13148 13187 1 3 SEQ ID NO: 3908 taagattaaatatttactt 11277 11298 SEQ ID NO: 5251 ttaatctttcataagtcaa 8893 8612 1 3 SEQ ID NO: 3909 aaagattactttgagaaat 11332 11351 SEQ ID NO: 5252 ggacaaggcccagaatctg 13334 13363 1 3 SEQ ID NO: 3910 aggaaatagttggattacc 11432 11451 SEQ ID NO: 5253 attcttaacgggattcctt 13110 13129 1 3 SEQ ID NO: 3911 attttgatgatgctggccc 11453 11472 SEQ ID NO: 5254 aattgttgaaagaaaaact 12641 12660 1 3 SEQ ID NO: 3912 gaattatcttttaaacatt 11536 11555 SEQ ID NO: 5255 agcttgtttgccagtgggc 8525 8547 1 3 SEQ ID NO: 3913 ttaccaccagtttgaagtt 11591 11610 SEQ ID NO: 5256 ttaatctttcataagtcaa 12693 12712 1 3 SEQ ID NO: 3914 ttgcagtgtatcggacaga 11608 11627 SEQ ID NO: 5257 agcttgtttgccagtgggc 11833 11652 1 3 SEQ ID NO: 3915 cattcagaaccagggaaga 11639 11685 SEQ ID NO: 5258 tgaaatcattgaaaattta 9231 9550 1 3 SEQ ID NO: 3916 acaaactgtttatagtccc 11691 11710 SEQ ID NO: 5259 caggaggctttaagttgtc 12259 12278 1 3 SEQ ID NO: 3917 ggatttcaatacatttcac 11692 11711 SEQ ID NO: 5260 agcttgtttgccagtgggc 12258 12277 1 3 SEQ ID NO: 3918 ttgtagaaatgaaagtaaa 11703 11722 SEQ ID NO: 5261 taaagccattcaggtctct 13179 13196 1 3 SEQ ID NO: 3919 ctgaacagtgagctgcagt 11807 11826 SEQ ID NO: 5262 cttcatcgttcatttgagt 8504 8523 1 3 SEQ ID NO: 3920 aatccaatctcctctttgc 12004 12023 SEQ ID NO: 5263 cttatgggatttcctaagg 11438 11457 1 3 SEQ ID NO: 3921 atttgattttcaagcaaag 120456 12075 SEQ ID NO: 5264 cttcatcgttcatttgagt 13285 13305 1 3 SEQ ID NO: 3922 ttttgattttcaagcaaat 12060 12079 SEQ ID NO: 5265 ttccatcacaaatcctttg 13980 13979 1 3 SEQ ID NO: 3923 tgatttcaagcaaatgcag 12074 142093 SEQ ID NO: 5266 tctcaaagagttacaacag 13711 13730 1 3 SEQ ID NO: 3924 atgctgtttgggaaattgg 12264 12283 SEQ ID NO: 5267 tattgaaatatgatttttt 11667 11688 1 3 SEQ ID NO: 3925 ttgtagaaatgaaagtaaa 12300 12319 SEQ ID NO: 5268 aattgttgaaagaaaaact 3018 3037 1 3 SEQ ID NO: 3926 attttgatgatgctggccc 12327 12348 SEQ ID NO: 5269 ggacaaggcccagaatctg 6508 6527 1 3 SEQ ID NO: 3927 gaattatcttttaaacatt 12362 12381 SEQ ID NO: 5270 aaagaaacctatggcctta 12763 12782 1 3 SEQ ID NO: 3928 ttaccaccagtttgaagtt 12475 12494 SEQ ID NO: 5271 attcttaacgggattcctt 13115 13124 1 3 SEQ ID NO: 3929 ttgcagtgtatcggacaga 12584 12603 SEQ ID NO: 5272 taaagccattcaggtctct 12640 12669 1 3 SEQ ID NO: 3930 ggatttcaatacatttcac 12736 12755 SEQ ID NO: 5273 gacatgtttgaagggtgga 12865 12884 1 3 SEQ ID NO: 3931 ttgtagaaatgaaagtaaa 12877 12898 SEQ ID NO: 5274 tgggctaaacgtttaattg 13524 13543 1 3 SEQ ID NO: 3932 ctgaacagtgagctgcagt 12974 12993 SEQ ID NO: 5275 tattgaaatatgatttttt 5802 5821 1 3 SEQ ID NO: 3933 aatccaatctcctctttgc 13001 13020 SEQ ID NO: 5276 agcttgtttgccagtgggc 13964 13983 1 3 SEQ ID NO: 3934 atttgattttcaagcaaag 13065 13084 SEQ ID NO: 5277 aaagaaacctatggcctta 13200 13219 1 3 SEQ ID NO: 3935 ttttgattttcaagcaaat 13080 13099 SEQ ID NO: 5278 attcttaacgggattcctt 13615 13834 1 3 SEQ ID NO: 3936 tgatttcaagcaaatgcag 13150 13169 SEQ ID NO: 5279 taaagccattcaggtctct 9566 9585 1 3 SEQ ID NO: 3937 atgctgtttgggaaattgg 13180 13199 SEQ ID NO: 5280 gacatgtttgaagggtgga 11702 11721 1 3 SEQ ID NO: 3938 tgctgttttttggaaatgc 13276 13208 SEQ ID NO: 5281 tgggctaaacgtttaattg 13937 13956 1 3 SEQ ID NO: 3939 aaaaaaatacactggacgt 13311 13330 SEQ ID NO: 5282 tattgaaatatgatttttt 8295 8314 1 3 SEQ ID NO: 3940 ctgaaaaagttagtgaaag 13442 13481 SEQ ID NO: 5283 taaagccattcaggtctct 13834 13853 1 3 SEQ ID NO: 3941 tttttcccagacagtgtca 13509 13628 SEQ ID NO: 5284 attcttaacgggattcctt 13821 13840 1 3 SEQ ID NO: 3942 ttttcccagacagtgtcca 13570 13555 SEQ ID NO: 5285 taaagccattcaggtctct 7999 8018 1 3 SEQ ID NO: 3943 attttgatgatgctggccc 13680 13899 SEQ ID NO: 5286 gacatgtttgaagggtgga 10422 10441 1 3 SEQ ID NO: 3944 gaattatcttttaaacatt 13693 13712 SEQ ID NO: 5287 tgggctaaacgtttaattg 7370 7389 1 3 SEQ ID NO: 3945 ttaccaccagtttgaagtt 13811 13830 SEQ ID NO: 5288 tattgaaatatgatttttt 10382 10401 1 3 SEQ ID NO: 3946 ttgcagtgtatcggacaga 14043 14082 SEQ ID NO: 5289 agcttgtttgccagtgggc 7884 7883 1 3 SEQ ID NO: 3947 aaagattactttgagaaat 14057 14076 SEQ ID NO: 5290 attcttaacgggattcctt 9842 9661 1 3

TABLE 10 Selected palindromic sequences from human glucose-6-phosphatase Start End Start End Source Index Index Match Index Index # B SEQ ID NO: 5291 ggccattccagaagggaag 222 241 SEQ ID NO: 5369 cttccgttctgtaatggcc 1340 1359 1 6 SEQ ID NO: 5292 tgccatctcgagagttcca 223 242 SEQ ID NO: 5370 tggaactctctccatggca 1339 1358 1 6 SEQ ID NO: 5293 catgtcaaacactttgtta 417 436 SEQ ID NO: 5371 taacaaattccttgacatg 1492 1511 1 6 SEQ ID NO: 5294 tttgttataaatcttattg 418 437 SEQ ID NO: 5372 caataagatcaatagcaaa 1491 1510 1 6 SEQ ID NO: 5295 tctggaaaaagggtcatga 521 540 SEQ ID NO: 5373 tccatgtcccatttacaga 2945 2964 1 6 SEQ ID NO: 5296 cagctcttgttcaggtcca 1886 1905 SEQ ID NO: 5374 tggacctgcaccaaagctg 271 2750 1 6 SEQ ID NO: 5297 ggaggttccccagctctgc 1956 1975 SEQ ID NO: 5375 gcagccctgggaaaactcc 2983 3002 1 6 SEQ ID NO: 5298 ctgttttgaagactctcca 50 69 SEQ ID NO: 5376 tggagggtagtcataacag 2620 2639 1 5 SEQ ID NO: 5299 agtggctgaaacgtgtgca 51 70 SEQ ID NO: 5377 tgcagagctttctgccact 2619 2638 1 5 SEQ ID NO: 5300 ccaaaatagaagggaatct 487 506 SEQ ID NO: 5378 agattcctttctttttcaa 1295 1314 1 5 SEQ ID NO: 5301 tgaagagaagattgaattt 598 617 SEQ ID NO: 5379 aaattctcttttattttca 2982 3001 1 5 SEQ ID NO: 5302 agtggtggcaacaccagca 651 670 SEQ ID NO: 5380 tgctagtgaggccaacact 773 792 1 5 SEQ ID NO: 5303 aaggctccacaagtcatca 776 795 SEQ ID NO: 5381 tgatgatatctggaacctt 884 903 1 5 SEQ ID NO: 5304 gtccgccaggtttctagca 848 867 SEQ ID NO: 5382 tgctaagaaccttactgac 2107 2126 1 5 SEQ ID NO: 5305 tgatatctggaaccttgga 878 897 SEQ ID NO: 5383 ttcactgttcctgaaatca 2801 2620 1 5 SEQ ID NO: 5306 gtcaagttgagcaatttct 1439 1458 SEQ ID NO: 5384 agaaaaggcacaccttgac 1676 1896 1 5 SEQ ID NO: 5307 atccagatggaaaagggaa 1572 1591 SEQ ID NO: 5385 ttccaatttccctgtggat 2626 2645 1 5 SEQ ID NO: 5308 atttgtttgtcaaagaagt 1573 1592 SEQ ID NO: 5386 acttcagagagaaatacat 2625 2644 4 5 SEQ ID NO: 5309 ctggaaaatgtcagcctgg 1854 1873 SEQ ID NO: 5387 ccagacttcagttaccagc 2683 2702 2 5 SEQ ID NO: 5310 accaggaggttcttcttca 2509 2528 SEQ ID NO: 5388 tgaagtgtgatctcctggt 2996 3015 2 5 SEQ ID NO: 5311 aaagaagttctgaaaagat 0 19 SEQ ID NO: 5389 attccatcacaaaatcctt 612 831 2 4 SEQ ID NO: 5312 gctacagcttatggctcca 12 31 SEQ ID NO: 5390 tggatctaaatgcagtagc 1987 2006 2 4 SEQ ID NO: 5313 atcaatatgatcaatttgt 220 239 SEQ ID NO: 5391 caaagaaatcaagattgat 1440 1459 2 4 SEQ ID NO: 5314 gaattatctttaaaacatt 326 345 SEQ ID NO: 5392 atgtggacaaatataccgg 2426 2444 2 4 SEQ ID NO: 5315 cgaggcccgcgctgctggc 392 411 SEQ ID NO: 5393 gccagaagtgagatcctcg 782 801 1 4 SEQ ID NO: 5316 acaactatgaggctgagag 638 657 SEQ ID NO: 5394 ctctgagcaacaaattttt 1474 1493 1 4 SEQ ID NO: 5317 gctgagagttccagtggag 666 685 SEQ ID NO: 5395 ctccatggcaaatgtcagg 758 777 1 4 SEQ ID NO: 5318 tgaagaaaaaccaagaact 760 779 SEQ ID NO: 5396 gagtcattgaggttcttca 823 842 1 4 SEQ ID NO: 5319 cctacttacatcctgaaca 761 780 SEQ ID NO: 5397 tgttcataagggaggtagg 822 841 1 4 SEQ ID NO: 5320 ctacttacatcctgaacat 762 781 SEQ ID NO: 5398 atgttcataagggaggtag 821 840 1 4 SEQ ID NO: 5321 gagacagaagccaagagcc 767 786 SEQ ID NO: 5399 gcttggttttgcccagtct 2014 2033 1 4 SEQ ID NO: 5322 cactcactttaccgtcaag 862 881 SEQ ID NO: 5400 cttgaacaccaaagtcact 1687 1706 1 4 SEQ ID NO: 5323 ctgatcagcagcagccagt 863 882 SEQ ID NO: 5401 actgggaagtgcttatcag 1696 1705 1 4 SEQ ID NO: 5324 actggacgctaagaggaag 1028 1047 SEQ ID NO: 5402 cttccccaaagagaggagt 1663 1682 1 4 SEQ ID NO: 5325 agaggaagcatgtggcaga 1056 1075 SEQ ID NO: 5403 tctggcatttactttctct 2229 2248 1 4 SEQ ID NO: 5326 tgaagactctccaggaact 1217 1236 SEQ ID NO: 5404 agttgaaggagactattca 1446 1465 1 4 SEQ ID NO: 5327 ctctgagcaaaatatccag 1267 1286 SEQ ID NO: 5405 ctggttactgagctgagag 2311 2330 1 4 SEQ ID NO: 5328 atgaagcagtcacatctct 1598 1617 SEQ ID NO: 5406 agagctgccagtcttcatt 2967 2986 1 4 SEQ ID NO: 5329 ttgccacagctgattgagg 1764 1783 SEQ ID NO: 5407 cctcctacagtggtggcaa 2063 2062 1 4 SEQ ID NO: 5330 agctgattgaggtgtccag 1855 1874 SEQ ID NO: 5408 ctggattccacatgcagct 3003 3022 1 4 SEQ ID NO: 5331 tgctccactcacatcctcc 2215 2234 SEQ ID NO: 5409 ggaggctttaagttcagca 2617 2836 1 4 SEQ ID NO: 5332 tgaaacgtgtgcatgtcaa 2330 2349 SEQ ID NO: 5410 ttgggagagacaagtttca 3007 3026 1 4 SEQ ID NO: 5333 gacattgctaattacctga 2345 2364 SEQ ID NO: 5411 tcagaagctaagcaatgtc 2992 3011 1 4 SEQ ID NO: 5334 ttcttcttcagactttcct 197 216 SEQ ID NO: 5412 aggagagtccaaatttaga 1116 1135 1 3 SEQ ID NO: 5335 ccaatatcttgaaactcag 257 276 SEQ ID NO: 5413 tctgaattcattcaattgg 1446 1465 1 3 SEQ ID NO: 5336 aaagttagtgaaaagaagt 263 282 SEQ ID NO: 5414 aactaccctcactgccttt 423 442 1 3 SEQ ID NO: 5337 aagttagtgaaagaagttc 358 377 SEQ ID NO: 5415 gaacctctggcattttact 2548 2567 1 3 SEQ ID NO: 5338 aaagaagttctgaaagaat 464 483 SEQ ID NO: 5416 attctctggtaactacttt 2705 2724 1 3 SEQ ID NO: 5339 tttggctataccaaagatg 468 487 SEQ ID NO: 5417 catcttaggcactgacaaa 1419 1438 1 3 SEQ ID NO: 5340 tgttgagaagctgattaaa 492 511 SEQ ID NO: 5418 tttagccatcggctcaaca 2828 2647 1 3 SEQ ID NO: 5341 caggaagggctcaaagaat 563 572 SEQ ID NO: 5419 attcctttaacaattcctg 644 663 1 3 SEQ ID NO: 5342 aggaagggctcaaagaatg 564 583 SEQ ID NO: 5420 cattcctttaacaattcct 2036 2057 1 3 SEQ ID NO: 5343 gaagggctcaaagaatgac 572 591 SEQ ID NO: 5421 gtcagtcttcaggctcttc 2779 2798 1 3 SEQ ID NO: 5344 caaagaatgacttttttct 606 825 SEQ ID NO: 5422 agaaggatggcattttttg 2742 2761 1 3 SEQ ID NO: 5345 catggagaatgcctttgaa 645 864 SEQ ID NO: 5423 ttcagagccaaagtccatg 2185 2204 1 3 SEQ ID NO: 5346 ggagccaaggctggagtaa 650 669 SEQ ID NO: 5424 ttactccaacgccagctcc 1818 1837 1 3 SEQ ID NO: 5347 tcattccttccccaaagag 678 697 SEQ ID NO: 5425 ctctctggggcatctatgc 1950 1969 1 3 SEQ ID NO: 5348 acctatgagctccagagag 690 709 SEQ ID NO: 5426 ctctcaagaccacagaagg 2283 2302 1 3 SEQ ID NO: 5349 gggcaaaaacgtcttacag 691 710 SEQ ID NO: 5427 tctgaaagacaacgtgccc 2282 2301 1 3 SEQ ID NO: 5350 accctggaccttcagaaca 744 763 SEQ ID NO: 5428 tgttgctaaggttcagggt 2306 2325 1 3 SEQ ID NO: 5351 atgggcgacctaagttgtg 772 791 SEQ ID NO: 5429 cacaaattagtttcaccat 2713 2732 1 3 SEQ ID NO: 5352 gatgaagagaagattgaat 784 803 SEQ ID NO: 5430 atccagcttccccacactt 2805 2824 1 3 SEQ ID NO: 5354 caatgtgataccaaaaaaa 1004 1023 SEQ ID NO: 5431 tttttggaaatgccattgt 2405 2424 1 3 SEQ ID NO: 5355 gtagataccaaaaaaatga 1351 1370 SEQ ID NO: 5432 tcatgtgatgggtctctac 2847 2865 1 3 SEQ ID NO: 5356 gcttcagttcatttggact 1438 1457 SEQ ID NO: 5433 agtcaagaaggacttaagc 2750 2779 1 3 SEQ ID NO: 5357 tttgtttgtcaaagaagtc 1553 1572 SEQ ID NO: 5434 gacttcagagaaatacaaa 2297 2316 1 3 SEQ ID NO: 5358 ttgtttgtcaaagaagtca 1606 1625 SEQ ID NO: 5435 tgacttcagagaaatacaa 2131 2150 1 3 SEQ ID NO: 5359 tggcaatgggaaactcgct 1785 1804 SEQ ID NO: 5436 agcgagagtcccctgccat 2503 2522 1 3 SEQ ID NO: 5360 aacctctggcatttacttt 1786 1805 SEQ ID NO: 5437 aaaggagatgtcaagggtt 2502 2521 1 3 SEQ ID NO: 5361 catttactttctctcatga 1787 1806 SEQ ID NO: 5438 tcatttgaaagaataaatg 2501 2520 1 3 SEQ ID NO: 5362 aaagtcagtgccctgctta 1788 1807 SEQ ID NO: 5439 taagaaccttactgacttt 2500 2519 1 3 SEQ ID NO: 5363 tcccattttttgagacctt 1982 2001 SEQ ID NO: 5440 aaggacttcaggaatggga 2189 2208 1 3 SEQ ID NO: 5364 catcaatattgatcaattt 2081 2100 SEQ ID NO: 5441 aaattaaaaagtcttgatg 2319 2338 1 3 SEQ ID NO: 5365 taaagatagttatgattta 2086 2105 SEQ ID NO: 5442 taaaccaaaaacttggtta 2669 2908 1 3 SEQ ID NO: 5366 tattgatgaaatcattgaa 2231 2250 SEQ ID NO: 5443 ttcaaagacttaaaaaata 2601 2820 1 3 SEQ ID NO: 5367 atgatctacatttgtttat 2493 2512 SEQ ID NO: 5444 ataaagaaattaaagtcat 2555 2574 1 3 SEQ ID NO: 5368 agagacacatacagaatat 2519 2536 SEQ ID NO: 5445 atatattgtcagtgcctct 2977 2996 1 3 SEQ ID NO: 5369 gacacatacagaatataga 2652 2671 SEQ ID NO: 5446 tctaaattcagttcttgtc 2798 2817 1 3

TABLE 11 Selected palindromic sequences from human glucose-6-phosphatase Start End Start End Source Index Index Match Index Index # B SEQ ID NO: 5447 ggccattccagaagggaag 301 320 SEQ ID NO: 5471 cttccgttctgtaatggcc 598 617 1 6 SEQ ID NO: 5448 tgccatctcgagagttcca 831 850 SEQ ID NO: 5472 tggaactctctccatggca 859 878 1 6 SEQ ID NO: 5449 catgtcaaacactttgtta 879 898 SEQ ID NO: 5473 taacaaattccttgacatg 1019 1038 1 6 SEQ ID NO: 5450 tttgttataaatcttattg 376 395 SEQ ID NO: 5474 caataagatcaatagcaaa 1171 1190 1 5 SEQ ID NO: 5451 tctggaaaaagggtcatga 1478 1497 SEQ ID NO: 5475 tccatgtcccatttacaga 2057 2076 1 5 SEQ ID NO: 5452 cagctcttgttcaggtcca 2 21 SEQ ID NO: 5476 tggacctgcaccaaagctg 123 142 1 4 SEQ ID NO: 5453 ggaggttccccagctctgc 13 32 SEQ ID NO: 5477 gcagccctgggaaaactcc 66 85 1 4 SEQ ID NO: 5454 ctgttttgaagactctcca 51 70 SEQ ID NO: 5478 tggagggtagtcataacag 1448 1467 1 4 SEQ ID NO: 5455 agtggctgaaacgtgtgca 108 127 SEQ ID NO: 5479 tgcagagctttctgccact 2018 2037 1 4 SEQ ID NO: 5456 ccaaaatagaagggaatct 155 174 SEQ ID NO: 5480 agattcctttctttttcaa 1076 1095 1 4 SEQ ID NO: 5457 tgaagagaagattgaattt 156 175 SEQ ID NO: 5481 aaattctcttttattttca 1075 1094 1 4 SEQ ID NO: 5458 agtggtggcaacaccagca 177 196 SEQ ID NO: 5482 tgctagtgaggccaacact 1549 1568 1 4 SEQ ID NO: 5459 aaggctccacaagtcatca 325 344 SEQ ID NO: 5483 tgatgatatctggaacctt 1868 1887 1 4 SEQ ID NO: 5460 gtccgccaggtttctagca 1064 1083 SEQ ID NO: 5484 tgctaagaaccttactgac 2034 2053 1 4 SEQ ID NO: 5461 tgatatctggaaccttgga 1111 1130 SEQ ID NO: 5485 ttcactgttcctgaaatca 1659 1678 1 4 SEQ ID NO: 5462 gtcaagttgagcaatttct 1237 1256 SEQ ID NO: 5486 agaaaaggcacaccttgac 2201 2220 1 4 SEQ ID NO: 5463 atccagatggaaaagggaa 1545 1564 SEQ ID NO: 5487 ttccaatttccctgtggat 2121 2140 1 4 SEQ ID NO: 5464 atttgtttgtcaaagaagt 37 56 SEQ ID NO: 5488 acttcagagagaaatacat 724 743 1 3 SEQ ID NO: 5465 ctggaaaatgtcagcctgg 59 78 SEQ ID NO: 5489 ccagacttcagttaccagc 1124 1143 1 3 SEQ ID NO: 5466 accaggaggttcttcttca 132 151 SEQ ID NO: 5490 tgaagtgtgatctcctggt 1911 1930 1 3 SEQ ID NO: 5467 aaagaagttctgaaaagat 148 167 SEQ ID NO: 5491 attccatcacaaaatcctt 1748 1767 1 3 SEQ ID NO: 5468 gctacagcttatggctcca 194 213 SEQ ID NO: 5492 tggatctaaatgcagtagc 357 376 1 3 SEQ ID NO: 5469 atcaatatgatcaatttgt 296 315 SEQ ID NO: 5493 caaagaaatcaagattgat 518 537 1 3 SEQ ID NO: 5470 gaattatctttaaaacatt 966 985 SEQ ID NO: 5494 atgtggacaaatataccgg 1833 1852 1 3

TALBE 12 Selected palindromic sequences from human B-catein Start End Start End Source Index Index Match Index Index # B SEQ ID NO: 5495 ggccattccagaagggaag 70 89 SEQ ID NO: 5542 cttccgttctgtaatggcc 2152 2171 1 5 SEQ ID NO: 5496 tgccatctcgagagttcca 304 323 SEQ ID NO: 5543 tggaactctctccatggca 2387 2406 1 5 SEQ ID NO: 5497 catgtcaaacactttgtta 328 347 SEQ ID NO: 5544 taacaaattccttgacatg 985 1004 1 5 SEQ ID NO: 5498 tttgttataaatcttattg 334 353 SEQ ID NO: 5545 caataagatcaatagcaaa 791 810 1 5 SEQ ID NO: 5499 tctggaaaaagggtcatga 473 492 SEQ ID NO: 5546 tccatgtcccatttacaga 2037 2056 1 5 SEQ ID NO: 5500 cagctcttgttcaggtcca 677 696 SEQ ID NO: 5547 tggacctgcaccaaagctg 2539 2558 1 5 SEQ ID NO: 5501 ggaggttccccagctctgc 678 697 SEQ ID NO: 5548 gcagccctgggaaaactcc 2538 2557 1 5 SEQ ID NO: 5502 ctgttttgaagactctcca 383 402 SEQ ID NO: 5549 tggagggtagtcataacag 2176 2195 2 4 SEQ ID NO: 5503 agtggctgaaacgtgtgca 1839 1858 SEQ ID NO: 5550 tgcagagctttctgccact 2451 2470 2 4 SEQ ID NO: 5504 ccaaaatagaagggaatct 143 162 SEQ ID NO: 5551 agattcctttctttttcaa 1929 1946 1 4 SEQ ID NO: 5505 tgaagagaagattgaattt 151 170 SEQ ID NO: 5552 aaattctcttttattttca 1680 1699 1 4 SEQ ID NO: 5506 agtggtggcaacaccagca 260 279 SEQ ID NO: 5553 tgctagtgaggccaacact 2494 2513 1 4 SEQ ID NO: 5507 aaggctccacaagtcatca 383 402 SEQ ID NO: 5554 tgatgatatctggaacctt 1652 1671 1 4 SEQ ID NO: 5508 gtccgccaggtttctagca 384 403 SEQ ID NO: 5555 tgctaagaaccttactgac 2175 2194 1 4 SEQ ID NO: 5509 tgatatctggaaccttgga 454 473 SEQ ID NO: 5556 ttcactgttcctgaaatca 2517 2536 1 4 SEQ ID NO: 5510 gtcaagttgagcaatttct 563 582 SEQ ID NO: 5557 agaaaaggcacaccttgac 1652 1671 1 4 SEQ ID NO: 5511 atccagatggaaaagggaa 623 642 SEQ ID NO: 5558 ttccaatttccctgtggat 1820 1839 1 5 SEQ ID NO: 5512 atttgtttgtcaaagaagt 718 737 SEQ ID NO: 5559 acttcagagagaaatacat 1125 1145 1 5 SEQ ID NO: 5513 ctggaaaatgtcagcctgg 915 934 SEQ ID NO: 5560 ccagacttcagttaccagc 2029 2048 1 5 SEQ ID NO: 5514 accaggaggttcttcttca 1291 1310 SEQ ID NO: 5561 tgaagtgtgatctcctggt 2502 2521 1 5 SEQ ID NO: 5515 aaagaagttctgaaaagat 1356 1375 SEQ ID NO: 5562 attccatcacaaaatcctt 2162 2181 1 4 SEQ ID NO: 5516 gctacagcttatggctcca 1549 1568 SEQ ID NO: 5563 tggatctaaatgcagtagc 1605 1624 1 4 SEQ ID NO: 5517 atcaatatgatcaatttgt 2107 2126 SEQ ID NO: 5564 caaagaaatcaagattgat 2134 2153 1 4 SEQ ID NO: 5518 gaattatctttaaaacatt 245 264 SEQ ID NO: 5565 atgtggacaaatataccgg 828 847 2 3 SEQ ID NO: 5519 cgaggcccgcgctgctggc 4 23 SEQ ID NO: 5566 gccagaagtgagatcctcg 2420 2439 1 3 SEQ ID NO: 5520 acaactatgaggctgagag 60 79 SEQ ID NO: 5567 ctctgagcaacaaattttt 359 378 1 3 SEQ ID NO: 5521 gctgagagttccagtggag 174 193 SEQ ID NO: 5568 ctccatggcaaatgtcagg 437 456 1 3 SEQ ID NO: 5522 tgaagaaaaaccaagaact 213 232 SEQ ID NO: 5569 gagtcattgaggttcttca 2500 2519 1 3 SEQ ID NO: 5523 cctacttacatcctgaaca 245 264 SEQ ID NO: 5570 tgttcataagggaggtagg 275 294 1 3 SEQ ID NO: 5524 ctacttacatcctgaacat 323 342 SEQ ID NO: 5571 atgttcataagggaggtag 1579 1598 1 3 SEQ ID NO: 5525 gagacagaagccaagagcc 369 368 SEQ ID NO: 5572 gcttggttttgcccagtct 1972 1991 1 3 SEQ ID NO: 5526 cactcactttaccgtcaag 424 443 SEQ ID NO: 5573 cttgaacaccaaagtcact 2514 2533 1 3 SEQ ID NO: 5527 ctgatcagcagcagccagt 469 488 SEQ ID NO: 5574 actgggaagtgcttatcag 1276 1295 1 3 SEQ ID NO: 5528 actggacgctaagaggaag 516 535 SEQ ID NO: 5575 cttccccaaagagaggagt 1271 1290 1 3 SEQ ID NO: 5529 agaggaagcatgtggcaga 646 664 SEQ ID NO: 5576 tctggcatttactttctct 1430 1449 1 3 SEQ ID NO: 5530 tgaagactctccaggaact 646 665 SEQ ID NO: 5577 agttgaaggagactattca 1429 1448 1 3 SEQ ID NO: 5531 ctctgagcaaaatatccag 846 865 SEQ ID NO: 5578 ctggttactgagctgagag 1743 1762 1 3 SEQ ID NO: 5532 atgaagcagtcacatctct 974 993 SEQ ID NO: 5579 agagctgccagtcttcatt 2542 2561 1 3 SEQ ID NO: 5533 ttgccacagctgattgagg 1222 1241 SEQ ID NO: 5580 cctcctacagtggtggcaa 2037 2056 1 3 SEQ ID NO: 5534 agctgattgaggtgtccag 1347 1366 SEQ ID NO: 5581 ctggattccacatgcagct 1743 1762 1 3 SEQ ID NO: 5535 tgctccactcacatcctcc 1435 1454 SEQ ID NO: 5582 ggaggctttaagttcagca 1562 1581 1 3 SEQ ID NO: 5536 tgaaacgtgtgcatgtcaa 1839 1858 SEQ ID NO: 5583 ttgggagagacaagtttca 2370 2389 1 3 SEQ ID NO: 5537 gacattgctaattacctga 1852 1871 SEQ ID NO: 5584 tcagaagctaagcaatgtc 2053 2072 1 3 SEQ ID NO: 5538 ttcttcttcagactttcct 1915 1934 SEQ ID NO: 5585 aggagagtccaaatttaga 2065 2074 1 3 SEQ ID NO: 5539 ccaatatcttgaaactcag 1962 1981 SEQ ID NO: 5586 tctgaattcattcaattgg 2553 2572 1 3 SEQ ID NO: 5540 aaagttagtgaaaagaagt 2084 2103 SEQ ID NO: 5587 aactaccctcactgccttt 2114 2133 1 3 SEQ ID NO: 5541 aagttagtgaaagaagttc 2247 2256 SEQ ID NO: 5588 gaacctctggcattttact 2421 2440 1 3

TABLE 13 Selected palindromic sequences from human hepatitis C virus (HCV) Start End Start End Source Index Index Match Index Index # B SEQ ID NO: 5589 ggccattccagaagggaag 5314 5333 SEQ ID NO: 6135 cttccgttctgtaatggcc 6196 6215 1 9 SEQ ID NO: 5590 tgccatctcgagagttcca 1682 1701 SEQ ID NO: 6136 tggaactctctccatggca 8202 8221 1 8 SEQ ID NO: 5591 catgtcaaacactttgtta 1049 1068 SEQ ID NO: 6137 taacaaattccttgacatg 6151 6170 1 7 SEQ ID NO: 5592 ttgttataaatcttaattg 1352 1371 SEQ ID NO: 6138 caataagatcaatagcaaa 6053 6072 1 7 SEQ ID NO: 5593 tctggaaaagggtcatgga 2048 2067 SEQ ID NO: 6139 tccatgtcccatttacaga 6871 6890 1 7 SEQ ID NO: 5594 cagctcttgttcaggtcca 2049 2088 SEQ ID NO: 6140 tggacctgcaccaaagctg 6870 6889 1 7 SEQ ID NO: 5595 ggaggttccccagactcgc 5556 5575 SEQ ID NO: 6141 gcagccctgggaaaactcc 9254 9273 1 7 SEQ ID NO: 5596 ctgttttgaagactctcca 5744 5763 SEQ ID NO: 6142 tggagggtagtcataacag 9291 6310 1 7 SEQ ID NO: 5597 agtggctgaaacgtgtgca 6189 6206 SEQ ID NO: 6143 tgcagagctttctgcccac 5832 5851 1 7 SEQ ID NO: 5598 ccaaaatagaagggaatct 6249 6268 SEQ ID NO: 6144 agattcctttgccttttgg 6830 6649 1 7 SEQ ID NO: 5599 tgaagagaagattgaattt 6250 6269 SEQ ID NO: 6145 aaattctcttttcttttca 6829 6848 1 7 SEQ ID NO: 5600 agtggtgccaataccagca 8216 8235 SEQ ID NO: 6146 tgctagtgaggccaacact 8634 8653 1 7 SEQ ID NO: 5601 aaggctccacaagtcatca 1430 1449 SEQ ID NO: 6147 tgatgatatctggaacctt 9019 9038 2 6 SEQ ID NO: 5602 gtcagccaggtttatagca 370 389 SEQ ID NO: 6148 tgctaagaaccttactgac 4115 4134 1 6 SEQ ID NO: 5603 tgatatctggaaccttgaa 419 436 SEQ ID NO: 6149 ttcactgttcctgaaatca 5734 5753 1 6 SEQ ID NO: 5604 gtcaagttgagcaatttct 584 603 SEQ ID NO: 6150 agaaaaggcacaccttgac 6374 6393 1 6 SEQ ID NO: 5605 atccagatggaaaagggaa 1265 1284 SEQ ID NO: 6151 ttccccatttccctgtggt 8465 8484 1 6 SEQ ID NO: 5606 atttgtttgtcaaagaagt 1508 1527 SEQ ID NO: 6152 acttcagagaaatacaaat 4759 4778 1 6 SEQ ID NO: 5607 ctggaaaatgtcagcctgg 1624 1643 SEQ ID NO: 6153 ccagacttccgtttcccag 2594 2613 1 6 SEQ ID NO: 5608 accaggaggttcttcttca 1897 1916 SEQ ID NO: 6154 tgaagtgtagtctcctggt 4115 4134 1 6 SEQ ID NO: 5609 aaagaagttctgaaagaat 2032 2051 SEQ ID NO: 6155 attccatcacaatcctttt 6537 8556 1 6 SEQ ID NO: 5610 gctacagcttatggctcca 2238 2257 SEQ ID NO: 6156 tggatctaaatgcagtagc 7610 7629 1 6 SEQ ID NO: 5611 atccatcttgatcaatttg 2288 2307 SEQ ID NO: 6157 caaagaagtcaagattgat 9207 9220 1 6 SEQ ID NO: 5612 gaattatcttttaaaacat 2613 2632 SEQ ID NO: 6158 atgtgttaaccaaatattc 6827 6846 1 6 SEQ ID NO: 5613 cgaggcccgcgctgctggc 2817 2836 SEQ ID NO: 6159 gccagaagtgagatcctcg 7763 7762 1 6 SEQ ID NO: 5614 acaactatgaggctgagag 2997 3016 SEQ ID NO: 6160 ctctgagcaacaaatttgt 5454 5473 1 6 SEQ ID NO: 5615 gctgagagttccagtggcg 3014 3033 SEQ ID NO: 6161 ctccatggcaaatgtcagc 5518 5537 1 6 SEQ ID NO: 5616 tgaagaaaaccaagaactc 3314 3333 SEQ ID NO: 6162 gagtcattgcggttcttca 7641 7680 1 6 SEQ ID NO: 5617 cctcttacatcctgaacaa 3324 3343 SEQ ID NO: 6163 tgttcataagggaggtcgg 3915 3934 1 6 SEQ ID NO: 5618 ctacttacatcctgaacat 3874 3893 SEQ ID NO: 6164 atgttcataagggcggtag 7439 7458 1 6 SEQ ID NO: 5619 gagacagaagaagccaagc 3875 3894 SEQ ID NO: 6165 gcttggttttgccagtctc 7438 7457 1 6 SEQ ID NO: 5620 cactcactttaccgtcaag 3876 3895 SEQ ID NO: 6166 cttgaacacaaagtcagtg 7631 7600 1 6 SEQ ID NO: 5621 ctgatcagcagcagccagt 3961 3980 SEQ ID NO: 6167 actgggaagtgcttatcag 7252 7271 1 6 SEQ ID NO: 5622 actggccgctaagaggaag 4174 4193 SEQ ID NO: 6168 cttccccaaagagaccagt 8155 8174 1 6 SEQ ID NO: 5623 agaggaagcatgtggcaga 4245 4264 SEQ ID NO: 6169 tctggcatttactttctct 4853 4872 1 6 SEQ ID NO: 5624 tgaagactctccaggaact 4501 4520 SEQ ID NO: 6170 agttgaaggagactattca 8334 8353 1 6 SEQ ID NO: 5625 ctctgagcaaaatatccag 5517 5536 SEQ ID NO: 6171 ctggttactgagctgagag 3015 3034 1 6 SEQ ID NO: 5626 atgaagcagtcacatctct 7821 7840 SEQ ID NO: 6172 agagctgccagtccttcat 8564 8583 1 6 SEQ ID NO: 5627 ttgccacagctgattgagg 767 788 SEQ ID NO: 6173 cctcctacagtggtggcaa 3674 3693 2 5 SEQ ID NO: 5628 agctgattgaggtgtccag 646 665 SEQ ID NO: 6174 ctggattccacatgcagct 7519 7538 2 5 SEQ ID NO: 5629 tgctccactcacatcctcc 750 789 SEQ ID NO: 6175 ggaggctttaagttcagca 7923 7942 2 5 SEQ ID NO: 5630 tgaagcgtgtgcatgccaa 767 786 SEQ ID NO: 6176 ttgggagagacaagtttca 1363 1402 2 5 SEQ ID NO: 5631 gacattgctaattacctga 1222 1241 SEQ ID NO: 6177 tcagaagctaagcaatgtc 9147 9166 2 5 SEQ ID NO: 5632 ttcttcttcagactttcct 178 197 SEQ ID NO: 6178 aggagagtccaaattagaa 7341 7360 1 5 SEQ ID NO: 5633 ccaatatcttgaactcaga 179 196 SEQ ID NO: 6179 tctgaattcattcaattgg 7340 7359 1 5 SEQ ID NO: 5634 aaagttggtgaaagaagtt 368 367 SEQ ID NO: 6180 aactaccctcactgccttt 9443 9462 1 5 SEQ ID NO: 5635 aagtagtgaaagaaagttc 385 404 SEQ ID NO: 6181 gaacctctggcatttactt 4100 4119 1 5 SEQ ID NO: 5636 aaagaagttctgaaagaat 418 437 SEQ ID NO: 6182 attctctggtaactacttt 4754 4773 1 5 SEQ ID NO: 5637 tttggctataccaaagatg 444 463 SEQ ID NO: 6183 catcttaggcactgacaaa 1267 1285 1 5 SEQ ID NO: 5638 tgttgagaagctgattaaa 450 469 SEQ ID NO: 6184 tttagccatcggctcaaca 3164 3183 1 5 SEQ ID NO: 5639 caggaagggctcaaagaat 460 479 SEQ ID NO: 6185 attcctttaacaggttcag 9296 9315 1 5 SEQ ID NO: 5640 aggaagggctcaaagaatg 657 676 SEQ ID NO: 6186 cattcctttaacaattcct 5783 5802 1 5 SEQ ID NO: 5641 gaagggctcaaagaatgac 658 677 SEQ ID NO: 6187 gtcagtcttcaggctcttc 5782 5801 1 5 SEQ ID NO: 5642 caaagaatgacttttttct 659 678 SEQ ID NO: 6188 agaaggatggcattttttg 5781 5800 1 5 SEQ ID NO: 5643 catggagaatgcctttgaa 715 734 SEQ ID NO: 6189 ttcagagccaaagtccatg 3774 3793 1 5 SEQ ID NO: 5644 ggagccacggatggagtaa 718 737 SEQ ID NO: 6190 ttatccaacgccagctccc 6020 6039 1 5 SEQ ID NO: 5645 tcattccttccccaaagag 751 770 SEQ ID NO: 6191 ctctcggggcatctatgaa 7922 7941 1 5 SEQ ID NO: 5646 acctatgagctccagagag 776 795 SEQ ID NO: 6192 ctctcaagaccacagaggt 6110 5129 1 5 SEQ ID NO: 5647 gggcaaacgtcttacagaa 943 962 SEQ ID NO: 6193 tctgaaagaaacgtgtgca 6498 6517 1 5 SEQ ID NO: 5648 accctggacattcagaaca 1019 1038 SEQ ID NO: 6194 tgttgctaaggttcagggt 1495 1514 1 5 SEQ ID NO: 5649 atgggcgacctaagttgtg 1020 1039 SEQ ID NO: 6195 cacaaattagttcaccatt 1494 1513 1 5 SEQ ID NO: 5650 gatgaagagagagttgagt 1021 1040 SEQ ID NO: 6196 attccagcttccccacatc 1493 1512 1 5 SEQ ID NO: 5651 caatgtagataccaaaaaa 1085 1104 SEQ ID NO: 6197 tttttggaaatgccattgc 4323 4342 1 5 SEQ ID NO: 5652 gtagataccaaaaaaatga 1098 1117 SEQ ID NO: 6198 tcatgtgatgggtctcacc 8092 8111 1 5 SEQ ID NO: 5653 gcttcagttcatttggact 1112 1131 SEQ ID NO: 6199 agtcaagaaggacttaagc 4576 4595 1 5 SEQ ID NO: 5654 tttgtttgttcaaagaagt 1128 1147 SEQ ID NO: 6200 gactcagagaaatacaaac 9164 9183 1 5 SEQ ID NO: 5655 ttgtttgtcaaagaagtca 1215 1234 SEQ ID NO: 6201 tgactcagagaaatacaac 6374 6393 1 5 SEQ ID NO: 5656 tggcaatgggaaactcgct 1265 1265 SEQ ID NO: 6202 agcgagaatcaccctgcca 6340 6359 1 5 SEQ ID NO: 5657 aacctctggcatttacttt 1331 1350 SEQ ID NO: 6203 aaaggagatgtcaagggtt 6048 6065 1 5 SEQ ID NO: 5658 catttactttctctcatga 1558 1577 SEQ ID NO: 6204 tcatttgaaagaataaatg 5838 5857 1 5 SEQ ID NO: 5659 aaagtcagtgccctgctta 1619 1638 SEQ ID NO: 6205 taagaacctactgaacttt 2313 2332 1 5 SEQ ID NO: 5660 tcccattttttgagacctt 1641 1660 SEQ ID NO: 6206 aaggacttcaggaatggga 8609 8628 1 5 SEQ ID NO: 5661 catcaatattgatcaattt 1685 1704 SEQ ID NO: 6207 aatattaaaagtcttgatg 4257 4276 1 5 SEQ ID NO: 5662 taaagatagttatgattta 1738 1757 SEQ ID NO: 6208 taaaccaaaacttggttta 5084 5103 1 5 SEQ ID NO: 5663 tattgatgaaatcattgaa 1894 1913 SEQ ID NO: 6209 ttcaaagacttaaaacaat 6343 6362 1 5 SEQ ID NO: 5664 atgatctacatttgtttat 2026 2045 SEQ ID NO: 6210 ataaagaaattgaatcaat 6919 6938 1 5 SEQ ID NO: 5665 agagacacatacagaatat 2030 2049 SEQ ID NO: 6211 atatattgtcagtgcctct 4202 4221 1 5 SEQ ID NO: 5666 gacacatacagaatataga 2101 2120 SEQ ID NO: 6212 tctaaattcagttcttgtc 3886 3905 1 5 SEQ ID NO: 5667 agcatgtcaaacatttgtc 2115 2134 SEQ ID NO: 6213 acaaagtcagtgccctgct 3137 3156 1 5 SEQ ID NO: 5668 ttttagaggaaaccaaggc 2235 2254 SEQ ID NO: 6214 cctttgtgtacaccccaaa 4011 4030 1 5 SEQ ID NO: 5669 ttttagaggaaaccaaggc 2354 2373 SEQ ID NO: 6215 gcctttgtgtacaccaaaa 3400 3419 1 5 SEQ ID NO: 5670 ggaagatagacttcctgaa 2474 2493 SEQ ID NO: 6216 ttcagaaatactgtttccg 8861 8880 1 5 SEQ ID NO: 5671 cactgtttctgagtcccag 2666 2585 SEQ ID NO: 6217 ctgggacctaccaagagtg 7834 7853 1 5 SEQ ID NO: 5672 cacaaatcctttggctgtg 2698 2717 SEQ ID NO: 6218 cacatttcaggattgtggg 7865 7884 1 5 SEQ ID NO: 5673 ttcctggatacactgttcc 2699 2718 SEQ ID NO: 6219 ggaactgttgactcaggaa 7864 7883 1 5 SEQ ID NO: 5674 gaaatctcaagctttctat 2700 2719 SEQ ID NO: 6220 agagccaggtccgagcttc 7863 7882 1 5 SEQ ID NO: 5675 tttcttcatcttcatctgt 2913 2932 SEQ ID NO: 6221 acagctgaaagagatgaaa 6402 6421 1 5 SEQ ID NO: 5676 tctaccgctaaaggagcag 2928 2947 SEQ ID NO: 6222 ctgcacgcttgaggtagaa 9376 9395 1 5 SEQ ID NO: 5677 ctaccgtcaaaggagcagt 3401 3420 SEQ ID NO: 6223 actgcacgctttgaggtag 8066 8085 1 5 SEQ ID NO: 5678 agggcctctttttcaccaa 3574 3593 SEQ ID NO: 6224 ttggccaggaagtggccct 4158 4177 1 5 SEQ ID NO: 5679 ttctccatccctgtaaaag 3575 3594 SEQ ID NO: 6225 cttttcaccaaacgggaga 4157 4176 1 5 SEQ ID NO: 5680 gaaaaccaaagcagattct 3695 3714 SEQ ID NO: 6226 ataaactgcaagattttct 8543 8562 1 5 SEQ ID NO: 5681 actcactcattgatttcct 3704 3723 SEQ ID NO: 6227 agaaaatcaggatctgagt 6342 8351 1 5 SEQ ID NO: 5682 taaactaatagatgtaatc 3705 3724 SEQ ID NO: 6228 gattaccaccagcggttta 6341 6360 1 5 SEQ ID NO: 5683 caaaagagcttcaggaagc 4325 4344 SEQ ID NO: 6229 cttcgtgaagaatattttg 4979 4998 1 5 SEQ ID NO: 5684 tggaataatgctcagtgtt 4347 4366 SEQ ID NO: 6230 aacacttcattgaattcca 5804 5823 1 5 SEQ ID NO: 5685 gatttgaaatccaaaggag 4361 4380 SEQ ID NO: 6231 cttcagagaatacaaaatc 7443 7462 1 5 SEQ ID NO: 5686 atttgaaatccaaagaagt 4485 4508 SEQ ID NO: 6232 acttcagcgaaatacaaat 5806 5825 1 5 SEQ ID NO: 5687 atcaacagccgcttctttg 5165 5184 SEQ ID NO: 6233 caaagaagtcaagattgat 7225 7244 1 5 SEQ ID NO: 5688 tgttttgaagactctccag 5322 5341 SEQ ID NO: 6234 ctggaaagttaaaacaaca 8765 8784 1 5 SEQ ID NO: 5689 cccttctgatagatgtggt 5390 5409 SEQ ID NO: 6235 accaaagctggcaccaggg 7713 7732 1 5 SEQ ID NO: 5690 tgagcaagtgaagaacttt 5515 5534 SEQ ID NO: 6236 aaagccattcagtctctca 6550 6569 1 5 SEQ ID NO: 5691 atttgaaatccaaagaagt 5592 5611 SEQ ID NO: 6237 acttttctaaacttgaaat 8176 8194 1 5 SEQ ID NO: 5692 atccaaagaagtcccggaa 5603 5622 SEQ ID NO: 6238 ttccggggaaacctgggat 7633 7652 1 5 SEQ ID NO: 5693 agagcctacctccgcatct 5739 5758 SEQ ID NO: 6239 agatggtacgttagcctct 5895 5914 1 5 SEQ ID NO: 5694 aatgcctttgaaactcccc 6306 6325 SEQ ID NO: 6240 tgggaactacaatttcatt 8363 8382 1 5 SEQ ID NO: 5695 gaagtccaaattccggatt 6456 6475 SEQ ID NO: 6241 aatcttcaatttattcttc 4158 4177 1 5 SEQ ID NO: 5696 tgcaagcagaagccagaag 6488 6507 SEQ ID NO: 6242 cttcaggtttccatcgtga 8708 8727 1 5 SEQ ID NO: 5697 gaagagaagattgaatttg 6565 6584 SEQ ID NO: 6243 caaaacctactgtctcctc 8500 8519 1 5 SEQ ID NO: 5698 atgctaaaggcacatatgg 6967 6986 SEQ ID NO: 6244 ccatatgaaagtcaagcat 3814 3833 1 5 SEQ ID NO: 5699 tccctcacctccacctctg 7078 7097 SEQ ID NO: 6245 cagattctcgatgagggat 7278 7297 1 5 SEQ ID NO: 5700 atttacagctctgacaagt 7138 7157 SEQ ID NO: 6246 acttttctaaacttgaatt 9326 9345 1 5 SEQ ID NO: 5701 aggagcctaccaaaattat 7202 7221 SEQ ID NO: 6247 attatgttgaaaacagttc 9150 9169 1 5 SEQ ID NO: 5702 aaagctgaagcacatcaat 7301 7320 SEQ ID NO: 6248 attgttgctcatctccttt 9162 9181 1 5 SEQ ID NO: 5703 ctgctggaaacaacgagaa 7520 7539 SEQ ID NO: 6249 tttctgataccacccacag 645 664 1 5 SEQ ID NO: 5704 ttgaaggaattcttgaaaa 7606 7625 SEQ ID NO: 6250 ttttaaaagaaaatctcaa 4809 4828 1 5 SEQ ID NO: 5705 gaagtaaaagaaatttggg 7626 7645 SEQ ID NO: 6251 caaaaacctactgtctggg 8996 9015 1 5 SEQ ID NO: 5706 tgaagaagatggcaaattt 7627 7646 SEQ ID NO: 6252 aaatgtcagctcttgttca 8995 9014 1 5 SEQ ID NO: 5707 aggatctgagttattttgc 8635 8664 SEQ ID NO: 6253 gcaagtcagcccagttcct 8920 8939 1 5 SEQ ID NO: 5708 gtgcccttctcggttgctg 27 46 SEQ ID NO: 6254 cagccattgacatgagcac 785 804 2 4 SEQ ID NO: 5709 ggcgctgcctgcgctgctg 1206 1225 SEQ ID NO: 6255 cagctccaccgactccgcc 8459 8478 2 4 SEQ ID NO: 5710 ctgcgctgctgctgctgct 1624 1643 SEQ ID NO: 6256 agcagaaggtgcaagcagg 5847 5806 2 4 SEQ ID NO: 5711 gctggtggcgggcgccagg 2238 2257 SEQ ID NO: 6257 cctggattccacatgcagc 7701 7720 2 4 SEQ ID NO: 5712 aagaggaaatgctggaaaa 3322 3341 SEQ ID NO: 6258 ttttcttcactactacttc 5795 5814 2 4 SEQ ID NO: 5713 ctggaaaatgtcagcctgg 3324 3343 SEQ ID NO: 6259 ccagacttccacatcccag 5076 5095 2 4 SEQ ID NO: 5714 tggagtccctgggactgct 8366 3385 SEQ ID NO: 6260 agcatgcctagtttctcca 6545 6564 2 4 SEQ ID NO: 5715 ggagctcccgggacttgtg 3674 3593 SEQ ID NO: 6261 cagtacgcctagtttctcc 7718 7737 2 4 SEQ ID NO: 5716 tgggactgctgattcaaga 6189 6208 SEQ ID NO: 6262 tcttccatcacttggaaac 6382 6401 2 4 SEQ ID NO: 5717 ctgctgattcaagaagtgc 6967 6986 SEQ ID NO: 6263 gcacaccttgacattgaca 9147 9166 2 4 SEQ ID NO: 5718 tgccaccaggatcaactgc 1 20 SEQ ID NO: 6264 gcaggctgaactggtggca 550 569 1 4 SEQ ID NO: 5719 gccaccaggatcaactgca 28 47 SEQ ID NO: 6265 tgcaggctgaactggttgc 784 803 1 4 SEQ ID NO: 5720 tgcaaggttgagctggagg 95 114 SEQ ID NO: 6266 cctccacctctgatctgca 2946 2965 1 4 SEQ ID NO: 5721 cataggttgagctggaggt 104 123 SEQ ID NO: 6267 aaccctacatggaaagctt 6403 6422 1 4 SEQ ID NO: 5722 ctctgcagtccatccctga 105 124 SEQ ID NO: 6268 tcaggaagcttctcaagag 6402 6421 1 4 SEQ ID NO: 5723 cagcttcatcctgaagacc 113 132 SEQ ID NO: 6269 ggtcttgagttaaatgctg 9255 9274 1 4 SEQ ID NO: 5724 gcttcatcctgaatgacag 118 137 SEQ ID NO: 6270 ctggacgctaagaggaagc 6368 6387 1 4 SEQ ID NO: 5725 tcatcctgaagaccagcca 121 140 SEQ ID NO: 6271 tggcatggcattatgatcc 598 617 1 4 SEQ ID NO: 5726 gaaaaccaagaactctgag 243 262 SEQ ID NO: 6272 ctcaaccttaatgattttc 3953 3972 1 4 SEQ ID NO: 5727 agaactctgaggagtttgc 251 270 SEQ ID NO: 6273 gcaagctctacagtattct 3656 3675 1 4 SEQ ID NO: 5728 tctgaggagtttgccgggt 299 318 SEQ ID NO: 6274 ctgcaggggatcccccaga 2139 2158 1 4 SEQ ID NO: 5729 tttgctgcagccatgtcca 306 325 SEQ ID NO: 6275 tggaagtgcagtggcaaat 671 690 1 4 SEQ ID NO: 5730 caagaggggcatcatttct 331 350 SEQ ID NO: 6276 agaataaatgacgttcttg 2437 2456 1 4 SEQ ID NO: 5731 tcactttaccgtcaagacg 412 431 SEQ ID NO: 6277 cgtctacactatagaaatt 997 1016 1 4 SEQ ID NO: 5732 tttaccgtcaagacgaagg 451 470 SEQ ID NO: 6278 tccttgaccatgttgataa 6549 6568 1 4 SEQ ID NO: 5733 cactggacgctaagaaaga 511 530 SEQ ID NO: 6279 ttccagaaagcagccagtg 3891 3910 1 4 SEQ ID NO: 5734 aggaagcatgtggcagaag 512 531 SEQ ID NO: 6280 cttcatacacattatcctt 3890 3909 1 4 SEQ ID NO: 5735 caaggagcaacacctcttc 526 547 SEQ ID NO: 6281 gaagtagtactgcatcttg 553 572 1 4 SEQ ID NO: 5736 acagactttgaaacttgaa 531 550 SEQ ID NO: 6282 ttcaatcttcaatgctgtt 5416 5437 1 4 SEQ ID NO: 5737 tgatgaagcagtcacatct 532 551 SEQ ID NO: 6283 agatttgaggattccatca 5417 5436 1 4 SEQ ID NO: 5738 agcagtcacatctctcttg 577 596 SEQ ID NO: 6284 caaggagaaaactgactgc 8067 8086 1 4 SEQ ID NO: 5739 cagccccatcactttacat 609 628 SEQ ID NO: 6285 tgtagtctcctggtgctgg 706 725 1 4 SEQ ID NO: 5740 ctccactcatccttcagtg 630 649 SEQ ID NO: 6286 caggagcttagtaatggag 8523 8542 1 4 SEQ ID NO: 5741 catgcccaccccctcctgg 661 680 SEQ ID NO: 6287 tcagatgagggaacacatg 4064 4083 1 4 SEQ ID NO: 5742 gagagatcttcaacatggc 662 681 SEQ ID NO: 6288 gccaccctggaaactctct 4063 4082 1 4 SEQ ID NO: 5743 tcaaacatggcgacgcatc 724 743 SEQ ID NO: 6289 tgatcccacctctcattga 6472 6491 1 4 SEQ ID NO: 5744 ccaccttgtatgcgctgag 725 744 SEQ ID NO: 6290 ctcagggatctgaggggtg 64171 6490 1 4 SEQ ID NO: 5745 gtcaaacaactatatcata 764 783 SEQ ID NO: 6291 tcttgagttaaatgctgac 7414 7433 1 4 SEQ ID NO: 5746 tggacattgctaattacct 792 811 SEQ ID NO: 6292 aggtatattcgaaagtcca 5188 5207 1 4 SEQ ID NO: 5747 ggacattgctaattacctg 858 887 SEQ ID NO: 6293 caggtatattcgaaagtcc 5863 5882 1 4 SEQ ID NO: 5748 ttctgcgggtcattggaaa 944 963 SEQ ID NO: 6294 tttcacactgcccaagaaa 6847 6866 1 4 SEQ ID NO: 5749 ccagaactcaagtcttcaa 983 1002 SEQ ID NO: 6295 ttgaagtgtgtctcctggc 5712 5731 1 4 SEQ ID NO: 5750 agtcttcaattcctgaagt 1096 1115 SEQ ID NO: 6296 catttctgattggtggact 4592 4611 1 4 SEQ ID NO: 5751 tgagcaagtgaagaaactt 1207 1226 SEQ ID NO: 6297 aaagtgccactttttacta 8793 8612 1 4 SEQ ID NO: 5752 agcaagtgaaagaacttgt 1222 1241 SEQ ID NO: 6298 acaaagtcagttgcctggt 3814 3833 1 4 SEQ ID NO: 5753 tctgaaaagaatctcaact 1293 1312 SEQ ID NO: 6299 aagtccataatggttcaga 5981 6000 1 4 SEQ ID NO: 5754 actgtcatggacttcagaa 1352 1381 SEQ ID NO: 6300 ttctgaatatattgtcagt 6279 6298 1 4 SEQ ID NO: 5755 acttgacccagcctcagcc 1365 1385 SEQ ID NO: 6301 ggctccccctgagagaagt 5560 5579 1 4 SEQ ID NO: 5756 tccaaataactaccttcct 1381 1400 SEQ ID NO: 6302 aggaagatagaagatggat 5127 5146 1 4 SEQ ID NO: 5757 actaccctcactgcctttg 1387 1406 SEQ ID NO: 6303 caaatttgtggagggtagt 8641 8660 1 4 SEQ ID NO: 5758 ttggatttgcttcagctga 1430 1449 SEQ ID NO: 6304 tcagtataaatacaccaaa 6846 6865 1 4 SEQ ID NO: 5759 ttggaagctcttgggaaat 1454 1473 SEQ ID NO: 6305 tcccgattcacgcttccaa 7665 7684 1 4 SEQ ID NO: 5760 ggaagctctttttgggaag 1500 1519 SEQ ID NO: 6306 cttcagaaagctaccttcc 5530 5549 1 4 SEQ ID NO: 5761 tttttcccagacagtgtca 1512 1531 SEQ ID NO: 6307 tgacctctctaagccaaat 2278 2297 1 4 SEQ ID NO: 5762 agacagtgtcaacaagctt 1564 1583 SEQ ID NO: 6308 agctggttttgcccagttt 8393 8412 1 4 SEQ ID NO: 5763 cttggctataccaaagatt 1586 1605 SEQ ID NO: 6309 atctcgtgtctaggaaaag 2132 2151 1 4 SEQ ID NO: 5764 caaagatgataaaacatgg 1587 1606 SEQ ID NO: 6310 ctcaaggataacgtgtttg 2131 2160 1 4 SEQ ID NO: 5765 gatatggtaaatggaataa 1749 1768 SEQ ID NO: 6311 tttacttagggttaatttg 4176 4195 1 4 SEQ ID NO: 5766 ggaataatgctcagtgttg 1764 1783 SEQ ID NO: 6312 caacacttacttgaattcc 9063 9062 1 4 SEQ ID NO: 5767 tttgaaatccaaagaaatc 1844 1863 SEQ ID NO: 6313 gacttcagagaaatacaat 8807 8836 1 4 SEQ ID NO: 5768 cagatgattggagaggtca 1850 1869 SEQ ID NO: 6314 tccaatttcctgtgggtgg 7949 7968 1 4 SEQ ID NO: 5769 agaatgacttttttcttca 1866 1885 SEQ ID NO: 6315 tgaccacacaaacagtgct 6887 6906 1 4 SEQ ID NO: 5770 agaatgactttttcttcac 1831 1900 SEQ ID NO: 6316 tgaagtccggcttctttct 2295 2314 1 4 SEQ ID NO: 5771 gaactcccactggagctgc 1972 1991 SEQ ID NO: 6317 cagctcaaccgtacagttc 9241 9260 1 4 SEQ ID NO: 5772 atatctaaggtggtccaca 1982 2001 SEQ ID NO: 6318 tgacttcagtgcagaatat 7287 7306 1 4 SEQ ID NO: 5773 gctgaagtttatcattcct 1983 2002 SEQ ID NO: 6319 tggccccgtttaccataga 7286 7305 1 4 SEQ ID NO: 5774 attccttcccccaaagaga 2043 2062 SEQ ID NO: 6320 aggaggctttaagttcagc 9376 9395 1 4 SEQ ID NO: 5775 ctcattgagaaacaggcag 2092 2111 SEQ ID NO: 6321 gtctcttcctccatggaat 5324 5343 1 4 SEQ ID NO: 5776 ctcattgagaacaggcagt 2093 2112 SEQ ID NO: 6322 actgactgcacgctttgag 5323 5342 1 4 SEQ ID NO: 5777 ttgagcagtattctgtcag 2100 2119 SEQ ID NO: 6323 gctgagaaagtgcttcctt 8616 8635 1 4 SEQ ID NO: 5778 accttgtccagtgaagtcc 2124 2143 SEQ ID NO: 6324 ggacggtactgtcccaggt 8201 8220 1 4 SEQ ID NO: 5779 cacgtgaagtccaaattcc 2142 2161 SEQ ID NO: 6325 ggaaggcagagtttactgg 3611 3630 1 4 SEQ ID NO: 5780 acattcagaaaagaaagcg 2287 2306 SEQ ID NO: 6326 atttcctaaagctggattg 8162 8161 1 4 SEQ ID NO: 5781 gaaaaatcaagggtgttat 2314 2333 SEQ ID NO: 6327 ataaactgcaaagattttt 3128 3147 1 4 SEQ ID NO: 5782 aaatcaagggtgttatttc 2317 2336 SEQ ID NO: 6328 gaaaacaatgcattagatt 3837 3858 1 4 SEQ ID NO: 5783 tggcattatgatgaagaga 2323 2342 SEQ ID NO: 6329 tctcccgtgtatatgccaa 6206 6225 1 4 SEQ ID NO: 5784 aagagaagattgaatttga 2383 2402 SEQ ID NO: 6330 tcaaaacctactgtctctt 6286 6305 1 4 SEQ ID NO: 5785 aaatgacttccaatttccc 2391 2410 SEQ ID NO: 6331 gggaactacaatttcattt 5564 5583 1 4 SEQ ID NO: 5786 atgacttccaattttccct 2419 2438 SEQ ID NO: 6332 caggctgattacgagtcat 3939 3948 1 4 SEQ ID NO: 5787 acttccaattttccctgtg 2460 2479 SEQ ID NO: 6333 ccacgaaaaatatggaagt 8875 8694 1 4 SEQ ID NO: 5788 agttgcaatgagctcatgg 2579 2598 SEQ ID NO: 6334 ccatcagttcagataaact 9241 9260 1 4 SEQ ID NO: 5789 tttgcaagaccacctcaat 2582 2601 SEQ ID NO: 6335 attgacctgtccattcaaa 5559 5578 1 4 SEQ ID NO: 5790 gaaggagttcaacctccag 2621 2640 SEQ ID NO: 6336 ctggaaatttgtcattccc 3214 3233 1 4 SEQ ID NO: 5791 acttccacatcccagaaaa 2786 2805 SEQ ID NO: 6337 ttttaacaaaaagtggtgg 7726 7745 1 4 SEQ ID NO: 5792 ctcttcttaaaaagcgatg 2824 2843 SEQ ID NO: 6338 catcactgccaaaggagag 4647 4666 1 4 SEQ ID NO: 5793 aaaagagagggcgggtcaa 2837 2856 SEQ ID NO: 6339 tgactactcagggaaattt 3539 3558 1 4 SEQ ID NO: 5794 ttcctttgccttttggtgg 2838 2857 SEQ ID NO: 6340 ccacaaacaatgaagggaa 3538 3557 1 4 SEQ ID NO: 5795 caagtctcggggattccat 2939 2958 SEQ ID NO: 6341 atggaaaaaaaacagggct 4327 4346 1 4 SEQ ID NO: 5796 aagtccctactttttacat 3201 3220 SEQ ID NO: 6342 atgggaagtataagaactt 8325 8344 1 4 SEQ ID NO: 5797 tgcctctcctgggtgtttc 3217 3236 SEQ ID NO: 6343 agaaaacaaccaaggcaaa 5221 5240 1 4 SEQ ID NO: 5798 accagcacagaccacccca 3233 3252 SEQ ID NO: 6344 tgaagtgtagtctcctggt 9338 9357 1 4 SEQ ID NO: 5799 ccagcacagaccatttcag 260 3279 SEQ ID NO: 6345 ctgaaatacaagcttctgg 7617 7636 1 4 SEQ ID NO: 5800 actatcatgtgatgggtct 3261 3280 SEQ ID NO: 6346 agacacctgatttttatat 7616 7635 1 4 SEQ ID NO: 5801 accacagatgtctgcttca 3297 3316 SEQ ID NO: 6347 tgaaggctgatttcttgtt 4804 4823 1 4 SEQ ID NO: 5802 ccacagatgtctgcttcag 3301 3320 SEQ ID NO: 6348 ctgagcaactttttttgtt 5794 5813 1 4 SEQ ID NO: 5803 tttggactccaaaaaagaa 316 3335 SEQ ID NO: 6349 tttctctcatgatacaaaa 3755 3774 1 4 SEQ ID NO: 5804 tcaaagaagtcaagattga 3378 3397 SEQ ID NO: 6350 tcaaggataacgtgtttga 4761 4780 1 4 SEQ ID NO: 5805 atgagaactacgagctgac 3499 3518 SEQ ID NO: 6351 gtcagatattggtgctcat 4222 4241 1 4 SEQ ID NO: 5806 ttaaatctgacaacattgg 3509 3528 SEQ ID NO: 6352 cattcatttgaaggggaac 7304 7323 1 4 SEQ ID NO: 5807 gaagtataagaactttgcc 3525 3644 SEQ ID NO: 6353 ggcaaatttgaaggacttc 7734 7753 1 4 SEQ ID NO: 5808 aagtataagaactttgcca 3626 3645 SEQ ID NO: 6354 tggcaaatttgaaggactt 7733 7752 1 4 SEQ ID NO: 5809 ttcttcagcctgtttctgg 3659 3678 SEQ ID NO: 6355 cagaatccagatacaagaa 9410 9429 1 4 SEQ ID NO: 5810 ctggatcactaaattccca 3660 3679 SEQ ID NO: 6356 tgggtcttccagaaggccg 9409 9426 1 4 SEQ ID NO: 5811 aaattaatagtggtgctca 3682 3701 SEQ ID NO: 6357 tgagaagccccagaatttt 7938 7957 1 4 SEQ ID NO: 5812 agtgcaacgaccaacttga 3711 3730 SEQ ID NO: 6358 tcaaattcctggattacac 7279 7298 1 4 SEQ ID NO: 5813 ctgggaagtgcttatcagg 3715 3734 SEQ ID NO: 6359 cctgaccttcacataccag 7826 7845 1 4 SEQ ID NO: 5814 gcaaaaacaaatttcaggg 3775 3791 SEQ ID NO: 6360 aagtcaaagaaattttgct 5682 5701 1 4 SEQ ID NO: 5815 aaaaaacatttcaacttca 3792 3811 SEQ ID NO: 6361 tgaagtaaaagaaattttt 6806 6825 1 4 SEQ ID NO: 5816 tcagtcaagaagaacttaa 3793 3812 SEQ ID NO: 6362 ttaaggaacttccattcta 8805 6824 1 4 SEQ ID NO: 5817 tcaaatgacatgatgggct 3822 3841 SEQ ID NO: 6363 agcccatcaatatattgaa 5585 5604 1 4 SEQ ID NO: 5818 cacaaaacagtctgaacaa 3928 3947 SEQ ID NO: 6364 tgttttcaactgccttgta 7717 7736 1 4 SEQ ID NO: 5819 tcttcaaaacttgaccaca 3947 3968 SEQ ID NO: 6365 tgttttcctattttcaaga 4504 4523 1 4 SEQ ID NO: 5820 caagtttttattaggcact 3948 3967 SEQ ID NO: 6366 agttatttgctaaagcttg 4826 4847 1 4 SEQ ID NO: 5821 tggtaactactttaccagg 4032 4051 SEQ ID NO: 6367 ctgttttagaggaaagccg 4993 5012 1 4 SEQ ID NO: 5822 aacagtgacctgaaataca 4136 4157 SEQ ID NO: 6368 tgtatagcaaattcctgtt 4377 4396 1 4 SEQ ID NO: 5823 gggaaactacggctagaac 4139 4158 SEQ ID NO: 6369 gttccttccagatttccgc 4376 4395 1 4 SEQ ID NO: 5824 aacaaacttgccatcctcc 4183 4202 SEQ ID NO: 6370 gagaacagctttcgtttgg 6119 6138 1 4 SEQ ID NO: 5825 tcagcaagctataaagcag 4193 4212 SEQ ID NO: 6371 ctgctaagaaccttactga 5833 5852 1 4 SEQ ID NO: 5826 gcagacactgttgctaagg 4215 4237 SEQ ID NO: 6372 cctttcaagcactgactgc 8668 8688 1 4 SEQ ID NO: 5827 tctggggagaacatactgg 4335 4354 SEQ ID NO: 6373 ccaggttttccacaaccga 9560 9579 1 4 SEQ ID NO: 5828 ttctctcatgattacaaag 4500 4519 SEQ ID NO: 6374 ctttttcaaccaacgggaa 5948 5967 1 4 SEQ ID NO: 5829 ctgagcagacaggcacctg 4526 4545 SEQ ID NO: 6375 caggaggctttaagttgtc 9589 9588 1 4 SEQ ID NO: 5830 caatttaaacaaacaagat 4577 4596 SEQ ID NO: 6376 attccttcctttacaattg 8682 8701 1 4 SEQ ID NO: 5831 tggacgaaactctggctac 4618 4637 SEQ ID NO: 6377 gtcagcccaggttcctcaa 8128 8147 1 4 SEQ ID NO: 5832 ctttttactcagtgaccca 4611 4830 SEQ ID NO: 6378 tgggctaaacgtttaattg 5992 6011 1 4 SEQ ID NO: 5833 tcattgtggctttagaaga 4857 4876 SEQ ID NO: 6379 atcttcataagttcaatga 5863 6882 1 4 SEQ ID NO: 5834 aaaaccaagatgttcactc 4869 4888 SEQ ID NO: 6380 gagtgaaatgctgtttttt 8067 8086 1 4 SEQ ID NO: 5835 aggaatcgacaaccattat 4922 4941 SEQ ID NO: 6381 taatgatttcaatgttcct 8662 8681 1 4 SEQ ID NO: 5836 tagttgtactggaaaaacg 4982 4981 SEQ ID NO: 6382 acgttagcctctatagact 9518 9537 1 4 SEQ ID NO: 5837 ggaaaactgatcagagaag 4983 4982 SEQ ID NO: 6383 cttttacaattcatttccc 9517 9536 1 4 SEQ ID NO: 5838 gaaaacgtacaggaccccc 5082 5101 SEQ ID NO: 6384 gctttctctttcacaaatg 6908 6927 1 4 SEQ ID NO: 5839 aaagctgaagcccatcaat 5140 5159 SEQ ID NO: 6385 atttgattagttggcaagg 7906 7925 1 4 SEQ ID NO: 5840 aagtctaagacctaggaga 5278 5297 SEQ ID NO: 6386 tattgatgttagagtgctt 9596 9615 1 4 SEQ ID NO: 5841 tgaagcacatcaatattga 5293 5312 SEQ ID NO: 6387 tcaaccttaatgattttaa 7885 7904 1 4 SEQ ID NO: 5842 atcaatattgatcaatttg 5294 5313 SEQ ID NO: 6388 caaagccatacatgatggg 7884 7903 1 4 SEQ ID NO: 5843 taatgattatctgaattca 5301 5320 SEQ ID NO: 6389 tgaaatcattgaaaattta 6676 6695 1 4 SEQ ID NO: 5844 gattatcgaattcattcat 5316 5336 SEQ ID NO: 6390 tgaaagtacgctgaagttc 7620 7639 1 4 SEQ ID NO: 5845 aattgggagagaagccttt 5383 5402 SEQ ID NO: 6391 aaacattcctttaccactt 6478 6497 1 4 SEQ ID NO: 5846 aaaatagctattgctaata 5429 5448 SEQ ID NO: 6392 tattgaaatatgatttttt 8383 8402 1 4 SEQ ID NO: 5847 aaaattaacaagtccgtat 5461 5480 SEQ ID NO: 6393 atcataggttgaggttttt 5924 5943 1 4 SEQ ID NO: 5848 ttgaaaaatatttgattta 5598 5617 SEQ ID NO: 6394 ttaatctttcataagtcaa 7240 7259 1 4 SEQ ID NO: 5849 agacatccagcacctagct 5645 5664 SEQ ID NO: 6395 tgggctaaacgtttaattg 8541 8580 1 4 SEQ ID NO: 5850 caattcatttgaaagaaat 5868 5687 SEQ ID NO: 6396 tattgaaatatgatttttt 5913 5932 1 4 SEQ ID NO: 5851 aggtttaaatggataattt 5736 5756 SEQ ID NO: 6397 aattgttgaaagaaaaact 5832 5851 1 4 SEQ ID NO: 5852 cagaagctaagcaatgtcc 5831 5850 SEQ ID NO: 6398 ggacaaggcccagaatctg 7948 7967 1 4 SEQ ID NO: 5853 taagattaaatatttactt 5877 5896 SEQ ID NO: 6399 aaagaaacctatggcctta 8142 8161 1 4 SEQ ID NO: 5854 aaagattactttgagaaat 5944 5963 SEQ ID NO: 6400 attcttaacgggattcctt 7463 7472 1 4 SEQ ID NO: 5855 aggaaatagttggattacc 6056 6075 SEQ ID NO: 6401 taaagccattcaggtctct 6969 6989 1 4 SEQ ID NO: 5856 attttgatgatgctggccc 6057 6076 SEQ ID NO: 6402 gacatgtttgaagggtgga 6968 6987 1 4 SEQ ID NO: 5857 gaattatcttttaaacatt 6074 6093 SEQ ID NO: 6403 tgggctaaacgtttaattg 7505 7524 1 4 SEQ ID NO: 5858 ttaccaccagtttgaagtt 6104 6123 SEQ ID NO: 6404 tattgaaatatgatttttt 2751 2770 1 4 SEQ ID NO: 5859 ttgcagtgtatcggacaga 6117 6136 SEQ ID NO: 6405 agcttgtttgccagtgggc 7531 7550 1 4 SEQ ID NO: 5860 cattcagaaccagggaaga 6199 6218 SEQ ID NO: 6406 tgaaatcattgaaaattta 7130 7149 1 4 SEQ ID NO: 5861 acaaactgtttatagtccc 6240 6259 SEQ ID NO: 6407 caggaggctttaagttgtc 7444 7463 1 4 SEQ ID NO: 5862 ggatttcaatacatttcac 6314 6333 SEQ ID NO: 6408 agcttgtttgccagtgggc 8541 8560 1 4 SEQ ID NO: 5863 ttgtagaaatgaaagtaaa 6338 6357 SEQ ID NO: 6409 taaagccattcaggtctct 7804 7823 1 4 SEQ ID NO: 5864 ctgaacagtgagctgcagt 6538 6557 SEQ ID NO: 6410 aattgttgaaagaaaaact 6613 6632 1 4 SEQ ID NO: 5865 aatccaatctcctctttgc 6616 6635 SEQ ID NO: 6411 taaagccattcaggtctct 7317 7336 1 4 SEQ ID NO: 5866 atttgattttcaagcaaag 6617 6636 SEQ ID NO: 6412 ttaatctttcataagtcaa 7455 7474 1 4 SEQ ID NO: 5867 ttttgattttcaagcaaat 6682 6701 SEQ ID NO: 6413 ggacaaggcccagaatctg 7059 7078 1 4 SEQ ID NO: 5868 tgatttcaagcaaatgcag 7152 7171 SEQ ID NO: 6414 attcttaacgggattcctt 8185 8184 1 4 SEQ ID NO: 5869 atgctgtttgggaaattgg 7239 7258 SEQ ID NO: 6415 aattgttgaaagaaaaact 5599 6618 1 4 SEQ ID NO: 5870 tgctgttttttggaaatgc 7295 7314 SEQ ID NO: 6416 agcttgtttgccagtgggc 8409 8428 1 4 SEQ ID NO: 5871 aaaaaaatacactggacgt 7327 7346 SEQ ID NO: 6417 ttaatctttcataagtcaa 9502 9521 1 4 SEQ ID NO: 5872 actggagcttagtaagggc 7328 7347 SEQ ID NO: 6418 agcttgtttgccagtgggc 9504 9523 1 4 SEQ ID NO: 5873 cttctggaaagagggtcat 7330 7349 SEQ ID NO: 6419 agcttgtttgccagtgggc 7770 7789 1 4 SEQ ID NO: 5874 ggaaaagggtcatggaaat 7351 7370 SEQ ID NO: 6420 attccttccttacaattgg 7390 7409 1 4 SEQ ID NO: 5875 gggcctgccccagatttct 7460 7479 SEQ ID NO: 6421 gagaacatttatggaggcc 7617 7638 1 4 SEQ ID NO: 5876 ttctcagataggggaacac 7667 7686 SEQ ID NO: 6422 gtgtcctcaagctgagggg 7686 7705 1 4 SEQ ID NO: 5877 gatgaaagaattaagggga 7792 7811 SEQ ID NO: 6423 attccagcttcccaactac 9238 9257 1 4 SEQ ID NO: 5878 cttggactgtcaaataagt 8028 8047 SEQ ID NO: 6424 cttatgggatttcctaagg 8931 8950 1 4 SEQ ID NO: 5879 gcatccacaaacaatggag 8029 8048 SEQ ID NO: 6425 cttcatcgttcatttgagt 8930 8949 1 4 SEQ ID NO: 5880 cacaaacaatgaagggaat 8204 8223 SEQ ID NO: 6426 cttatgggatttcctaagg 7860 7679 1 4 SEQ ID NO: 5881 ccaaaatttctctcggctt 8470 8489 SEQ ID NO: 6427 cttcatcgttcatttgagt 9091 9110 1 4 SEQ ID NO: 5882 caaattttctctgctaaaa 8766 8785 SEQ ID NO: 6428 ttccatcacaaatcctttg 9089 9108 1 4 SEQ ID NO: 5883 tctgctggaaacaacaacg 8876 8895 SEQ ID NO: 6429 tctcaaagagttacaacag 2459 2478 1 4 SEQ ID NO: 5884 cagaagctaagcaatgtcc 8976 8995 SEQ ID NO: 6430 tattgaaatatgatttttt 9052 9071 1 4 SEQ ID NO: 5885 taagattaaatatttactt 9090 9109 SEQ ID NO: 6431 aattgttgaaagaaaaact 8765 8784 1 4 SEQ ID NO: 5886 aaagattactttgagaaat 1624 1643 SEQ ID NO: 6432 ggacaaggcccagaatctg 5897 5916 3 3 SEQ ID NO: 5887 aggaaatagttggattacc 1 20 SEQ ID NO: 6433 aaagaaacctatggcctta 1391 1410 2 3 SEQ ID NO: 5888 attttgatgatgctggccc 520 539 SEQ ID NO: 6434 attcttaacgggattcctt 6918 6937 2 3 SEQ ID NO: 5889 gaattatcttttaaacatt 662 881 SEQ ID NO: 6435 taaagccattcaggtctct 4553 4572 2 3 SEQ ID NO: 5890 ttaccaccagtttgaagtt 983 1002 SEQ ID NO: 6436 gacatgtttgaagggtgga 8697 8716 2 3 SEQ ID NO: 5891 ttgcagtgtatcggacaga 1249 1266 SEQ ID NO: 6437 tgggctaaacgtttaattg 5330 5349 2 3 SEQ ID NO: 5892 cattcagaaccagggaaga 1637 1656 SEQ ID NO: 6438 tattgaaatatgatttttt 5599 5618 2 3 SEQ ID NO: 5893 acaaactgtttatagtccc 1830 1849 SEQ ID NO: 6439 agcttgtttgccagtgggc 8989 9008 2 3 SEQ ID NO: 5894 ggatttcaatacatttcac 2026 2045 SEQ ID NO: 6440 tgaaatcattgaaaattta 9156 9175 2 3 SEQ ID NO: 5895 ttgtagaaatgaaagtaaa 2264 2283 SEQ ID NO: 6441 caggaggctttaagttgtc 9405 9424 2 3 SEQ ID NO: 5896 ttaccaccagtttgaagtt 2383 2402 SEQ ID NO: 6442 agcttgtttgccagtgggc 7287 7288 2 3 SEQ ID NO: 5897 ttgcagtgtatcggacaga 2838 2857 SEQ ID NO: 6443 taaagccattcaggtctct 5654 5573 2 3 SEQ ID NO: 5898 cattcagaaccagggaaga 4489 4508 SEQ ID NO: 6444 aattgttgaaagaaaaact 8097 6116 2 3 SEQ ID NO: 5899 acaaactgtttatagtccc 5736 5755 SEQ ID NO: 6445 taaagccattcaggtctct 6382 6401 2 3 SEQ ID NO: 5900 ggatttcaatacatttcac 8635 8654 SEQ ID NO: 6446 ttaatctttcataagtcaa 9284 9303 2 3 SEQ ID NO: 5901 ttgtagaaatgaaagtaaa 24 43 SEQ ID NO: 6447 ggacaaggcccagaatctg 9111 9130 1 3 SEQ ID NO: 5902 ctgaacagtgagctgcagt 25 44 SEQ ID NO: 6448 attcttaacgggattcctt 5451 5470 1 3 SEQ ID NO: 5903 aatccaatctcctctttgc 27 48 SEQ ID NO: 6449 aattgttgaaagaaaaact 458 477 1 3 SEQ ID NO: 5904 atttgattttcaagcaaag 36 55 SEQ ID NO: 6450 agcttgtttgccagtgggc 809 826 1 3 SEQ ID NO: 5905 ttttgattttcaagcaaat 88 107 SEQ ID NO: 6451 ttaatctttcataagtcaa 5191 5210 1 3 SEQ ID NO: 5906 acaaactgtttatagtccc 100 119 SEQ ID NO: 6452 agcttgtttgccagtgggc 1879 1898 1 3 SEQ ID NO: 5907 ggatttcaatacatttcac 119 136 SEQ ID NO: 6453 agcttgtttgccagtgggc 645 664 1 3 SEQ ID NO: 5908 ttgtagaaatgaaagtaaa 131 150 SEQ ID NO: 6454 attccttccttacaattgg 8993 9012 1 3 SEQ ID NO: 5909 ttaccaccagtttgaagtt 134 153 SEQ ID NO: 6455 tgggctaaacgtttaattg 1046 1067 1 3 SEQ ID NO: 5910 ttgcagtgtatcggacaga 142 161 SEQ ID NO: 6456 tattgaaatatgatttttt 5038 5057 1 3 SEQ ID NO: 5911 cattcagaaccagggaaga 161 180 SEQ ID NO: 6457 agcttgtttgccagtgggc 1406 1425 1 3 SEQ ID NO: 5912 acaaactgtttatagtccc 188 207 SEQ ID NO: 6458 tgaaatcattgaaaattta 6304 6323 1 3 SEQ ID NO: 5913 ggatttcaatacatttcac 194 213 SEQ ID NO: 6459 caggaggctttaagttgtc 7134 7153 1 3 SEQ ID NO: 5914 ttgtagaaatgaaagtaaa 210 229 SEQ ID NO: 6460 agcttgtttgccagtgggc 7486 7505 1 3 SEQ ID NO: 5915 ccaaaatttctctcggctt 215 234 SEQ ID NO: 6461 taaagccattcaggtctct 1967 1986 1 3 SEQ ID NO: 5916 caaattttctctgctaaaa 216 235 SEQ ID NO: 6462 ggacaaggcccagaatctg 1966 1985 1 3 SEQ ID NO: 5917 tctgctggaaacaacaacg 221 240 SEQ ID NO: 6463 attcttaacgggattcctt 1891 1910 1 3 SEQ ID NO: 5918 cagaagctaagcaatgtcc 273 292 SEQ ID NO: 6464 aattgttgaaagaaaaact 7756 7775 1 3 SEQ ID NO: 5919 taagattaaatatttactt 274 293 SEQ ID NO: 6465 agcttgtttgccagtgggc 7755 7774 1 3 SEQ ID NO: 5920 aaagattactttgagaaat 282 301 SEQ ID NO: 6466 ttaatctttcataagtcaa 2385 2404 1 3 SEQ ID NO: 5921 aggaaatagttggattacc 291 310 SEQ ID NO: 6467 agcttgtttgccagtgggc 544 563 1 3 SEQ ID NO: 5922 attttgatgatgctggccc 307 326 SEQ ID NO: 6468 agcttgtttgccagtgggc 4780 4799 1 3 SEQ ID NO: 5923 gaattatcttttaaacatt 312 331 SEQ ID NO: 6469 attccttccttacaattgg 4122 4141 1 3 SEQ ID NO: 5924 ttaccaccagtttgaagtt 442 451 SEQ ID NO: 6470 agcttgtttgccagtgggc 1023 1042 1 3 SEQ ID NO: 5925 taagattaaatatttactt 443 462 SEQ ID NO: 6471 agcttgtttgccagtgggc 1022 1041 1 3 SEQ ID NO: 5926 aaagattactttgagaaat 445 464 SEQ ID NO: 6472 attccttccttacaattgg 8348 8367 1 3 SEQ ID NO: 5927 aggaaatagttggattacc 446 465 SEQ ID NO: 6473 tgggctaaacgtttaattg 8347 8366 1 3 SEQ ID NO: 5928 attttgatgatgctggccc 497 516 SEQ ID NO: 6474 tattgaaatatgatttttt 7922 7941 1 3 SEQ ID NO: 5929 gaattatcttttaaacatt 504 523 SEQ ID NO: 6475 agcttgtttgccagtgggc 3268 3287 1 3 SEQ ID NO: 5930 ttaccaccagtttgaagtt 505 524 SEQ ID NO: 6476 tgaaatcattgaaaattta 4749 4768 1 3 SEQ ID NO: 5931 ctgaaaaagttagtgaaag 520 539 SEQ ID NO: 6477 caggaggctttaagttgtc 774 793 1 3 SEQ ID NO: 5932 tttttcccagacagtgtca 527 546 SEQ ID NO: 6478 agcttgtttgccagtgggc 2252 2271 1 3 SEQ ID NO: 5933 ttttcccagacagtgtcca 554 573 SEQ ID NO: 6479 taaagccattcaggtctct 5020 5039 1 3 SEQ ID NO: 5934 cattcagaaccagggaaga 559 578 SEQ ID NO: 6480 cttcatcgttcatttgagt 4324 4343 1 3 SEQ ID NO: 5935 acaaactgtttatagtccc 561 580 SEQ ID NO: 6481 cttatgggatttcctaagg 4064 4083 1 3 SEQ ID NO: 5936 ggatttcaatacatttcac 564 583 SEQ ID NO: 6482 cttcatcgttcatttgagt 4162 4181 1 3 SEQ ID NO: 5937 ttgtagaaatgaaagtaaa 590 609 SEQ ID NO: 6483 ttccatcacaaatcctttg 727 746 1 3 SEQ ID NO: 5938 ccaaaatttctctcggctt 605 624 SEQ ID NO: 6484 tctcaaagagttacaacag 1180 1199 1 3 SEQ ID NO: 5939 caaattttctctgctaaaa 606 625 SEQ ID NO: 6485 tattgaaatatgatttttt 2370 2389 1 3 SEQ ID NO: 5940 tctgctggaaacaacaacg 622 641 SEQ ID NO: 6486 aattgttgaaagaaaaact 2459 2478 1 3 SEQ ID NO: 5941 cagaagctaagcaatgtcc 624 643 SEQ ID NO: 6487 ggacaaggcccagaatctg 4261 4280 1 3 SEQ ID NO: 5942 taagattaaatatttactt 633 652 SEQ ID NO: 6488 aaagaaacctatggcctta 3688 3707 1 3 SEQ ID NO: 5943 aaagattactttgagaaat 634 653 SEQ ID NO: 6489 attcttaacgggattcctt 3687 3706 1 3 SEQ ID NO: 5944 aggaaatagttggattacc 642 861 SEQ ID NO: 6490 taaagccattcaggtctct 2935 2954 1 3 SEQ ID NO: 5945 attttgatgatgctggccc 646 665 SEQ ID NO: 6491 gacatgtttgaagggtgga 4439 4458 1 3 SEQ ID NO: 5946 gaattatcttttaaacatt 711 730 SEQ ID NO: 6492 tgggctaaacgtttaattg 8839 8858 1 3 SEQ ID NO: 5947 ttaccaccagtttgaagtt 750 769 SEQ ID NO: 6493 tattgaaatatgatttttt 5625 5644 1 3 SEQ ID NO: 5948 taagattaaatatttactt 767 786 SEQ ID NO: 6494 agcttgtttgccagtgggc 898 817 1 3 SEQ ID NO: 5949 aaagattactttgagaaat 832 851 SEQ ID NO: 6495 tgaaatcattgaaaattta 3092 3111 1 3 SEQ ID NO: 5950 aggaaatagttggattacc 922 941 SEQ ID NO: 6496 caggaggctttaagttgtc 2751 2770 1 3 SEQ ID NO: 5951 attttgatgatgctggccc 968 987 SEQ ID NO: 6497 agcttgtttgccagtgggc 7512 7531 1 3 SEQ ID NO: 5952 aggtttaaatggataattt 1070 1080 SEQ ID NO: 6498 taaagccattcaggtctct 5763 5782 1 3 SEQ ID NO: 5953 cagaagctaagcaatgtcc 1080 1099 SEQ ID NO: 6499 aattgttgaaagaaaaact 5279 5298 1 3 SEQ ID NO: 5954 taagattaaatatttactt 1106 1125 SEQ ID NO: 6500 taaagccattcaggtctct 1888 1907 1 3 SEQ ID NO: 5955 aaagattactttgagaaat 1140 1159 SEQ ID NO: 6501 ttaatctttcataagtcaa 8921 8940 1 3 SEQ ID NO: 5956 aggaaatagttggattacc 1168 1187 SEQ ID NO: 6502 ggacaaggcccagaatctg 2615 2634 1 3 SEQ ID NO: 5957 attttgatgatgctggccc 1206 1225 SEQ ID NO: 6503 attcttaacgggattcctt 3779 3798 1 3 SEQ ID NO: 5958 gaattatcttttaaacatt 1208 1227 SEQ ID NO: 6504 aattgttgaaagaaaaact 9282 9301 1 3 SEQ ID NO: 5959 ttaccaccagtttgaagtt 1249 1268 SEQ ID NO: 6505 agcttgtttgccagtgggc 4809 44828 1 3 SEQ ID NO: 5960 ttgcagtgtatcggacaga 1254 1273 SEQ ID NO: 6506 ttaatctttcataagtcaa 3615 3634 1 3 SEQ ID NO: 5961 cattcagaaccagggaaga 1262 1281 SEQ ID NO: 6507 agcttgtttgccagtgggc 2766 2785 1 3 SEQ ID NO: 5962 acaaactgtttatagtccc 1270 1289 SEQ ID NO: 6508 tgaaatcattgaaaattta 1460 1479 1 3 SEQ ID NO: 5963 ggatttcaatacatttcac 1288 1307 SEQ ID NO: 6509 caggaggctttaagttgtc 3943 3962 1 3 SEQ ID NO: 5964 ttgtagaaatgaaagtaaa 1344 1363 SEQ ID NO: 6510 agcttgtttgccagtgggc 8593 8612 1 3 SEQ ID NO: 5965 ctgaacagtgagctgcagt 1360 1379 SEQ ID NO: 6511 taaagccattcaggtctct 5306 5325 1 3 SEQ ID NO: 5966 aatccaatctcctctttgc 1396 1415 SEQ ID NO: 6512 cttcatcgttcatttgagt 1695 1714 1 3 SEQ ID NO: 5967 atttgattttcaagcaaag 1408 1427 SEQ ID NO: 6513 cttatgggatttcctaagg 7865 7884 1 3 SEQ ID NO: 5968 ttttgattttcaagcaaat 1472 1491 SEQ ID NO: 6514 cttcatcgttcatttgagt 7886 7907 1 3 SEQ ID NO: 5969 tgatttcaagcaaatgcag 1492 1511 SEQ ID NO: 6515 ttccatcacaaatcctttg 4420 4439 1 3 SEQ ID NO: 5970 atgctgtttgggaaattgg 1504 1523 SEQ ID NO: 6516 tctcaaagagttacaacag 7440 7459 1 3 SEQ ID NO: 5971 tgctgttttttggaaatgc 1507 1526 SEQ ID NO: 6517 tattgaaatatgatttttt 3789 3808 1 3 SEQ ID NO: 5972 aaaaaaatacactggacgt 1517 1536 SEQ ID NO: 6518 aattgttgaaagaaaaact 1493 1512 1 3 SEQ ID NO: 5973 actggagcttagtaagggc 1571 1590 SEQ ID NO: 6519 ggacaaggcccagaatctg 5382 5401 1 3 SEQ ID NO: 5974 cttctggaaagagggtcat 1637 1656 SEQ ID NO: 6520 aaagaaacctatggcctta 4346 4365 1 3 SEQ ID NO: 5975 ggaaaagggtcatggaaat 1696 1715 SEQ ID NO: 6521 attcttaacgggattcctt 3859 3888 1 3 SEQ ID NO: 5976 gggcctgccccagatttct 1714 1733 SEQ ID NO: 6522 taaagccattcaggtctct 2667 2686 1 3 SEQ ID NO: 5977 ttctcagataggggaacac 1730 1749 SEQ ID NO: 6523 gacatgtttgaagggtgga 8673 8692 1 3 SEQ ID NO: 5978 gatgaaagaattaagggga 1746 1765 SEQ ID NO: 6524 tgggctaaacgtttaattg 2914 2933 1 3 SEQ ID NO: 5979 cttggactgtcaaataagt 1786 1805 SEQ ID NO: 6525 tattgaaatatgatttttt 3658 3677 1 3 SEQ ID NO: 5980 ggaaaagggtcatggaaat 1810 1829 SEQ ID NO: 6526 agcttgtttgccagtgggc 3264 3303 1 3 SEQ ID NO: 5981 gggcctgccccagatttct 1830 1849 SEQ ID NO: 6527 tgaaatcattgaaaattta 4748 4767 1 3 SEQ ID NO: 5982 cttggactgtcaaataagt 1837 1856 SEQ ID NO: 6528 tgaaatcattgaaaattta 6123 6142 1 3 SEQ ID NO: 5983 ggatttcaatacatttcac 1890 1909 SEQ ID NO: 6529 aaagaaacctatggcctta 4761 4780 1 3 SEQ ID NO: 5984 ttgtagaaatgaaagtaaa 1924 1943 SEQ ID NO: 6530 attcttaacgggattcctt 2320 2339 1 3 SEQ ID NO: 5985 ccaaaatttctctcggctt 1974 1993 SEQ ID NO: 6531 taaagccattcaggtctct 8596 8615 1 3 SEQ ID NO: 5986 caaattttctctgctaaaa 1975 1994 SEQ ID NO: 6532 gacatgtttgaagggtgga 8595 8614 1 3 SEQ ID NO: 5987 tctgctggaaacaacaacg 1996 2015 SEQ ID NO: 6533 tgggctaaacgtttaattg 8366 8365 1 3 SEQ ID NO: 5988 cagaagctaagcaatgtcc 1997 2016 SEQ ID NO: 6534 tattgaaatatgatttttt 7607 7626 1 3 SEQ ID NO: 5989 taagattaaatatttactt 2020 2039 SEQ ID NO: 6535 agcttgtttgccagtgggc 6740 5759 1 3 SEQ ID NO: 5990 aaagattactttgagaaat 2028 2047 SEQ ID NO: 6536 tgaaatcattgaaaattta 3512 3531 1 3 SEQ ID NO: 5991 aggaaatagttggattacc 2042 2061 SEQ ID NO: 6537 caggaggctttaagttgtc 9376 9396 1 3 SEQ ID NO: 5992 attttgatgatgctggccc 2071 2090 SEQ ID NO: 6538 agcttgtttgccagtgggc 8581 8580 1 3 SEQ ID NO: 5993 gaattatcttttaaacatt 2184 2203 SEQ ID NO: 6539 taaagccattcaggtctct 8600 8619 1 3 SEQ ID NO: 5994 ttaccaccagtttgaagtt 2201 2220 SEQ ID NO: 6540 aattgttgaaagaaaaact 6717 6736 1 3 SEQ ID NO: 5995 taagattaaatatttactt 2257 2276 SEQ ID NO: 6541 taaagccattcaggtctct 6411 6430 1 3 SEQ ID NO: 5996 aaagattactttgagaaat 2264 2283 SEQ ID NO: 6542 ttaatctttcataagtcaa 9009 9028 1 3 SEQ ID NO: 5997 aggaaatagttggattacc 2268 2287 SEQ ID NO: 6543 ggacaaggcccagaatctg 3236 3255 1 3 SEQ ID NO: 5998 attttgatgatgctggccc 2275 2294 SEQ ID NO: 6544 attcttaacgggattcctt 6761 6780 1 3 SEQ ID NO: 5999 gaattatcttttaaacatt 2309 2328 SEQ ID NO: 6545 aattgttgaaagaaaaact 3799 3818 1 3 SEQ ID NO: 6000 ttaccaccagtttgaagtt 2315 2334 SEQ ID NO: 6546 agcttgtttgccagtgggc 4357 4376 1 3 SEQ ID NO: 6001 ctgaaaaagttagtgaaag 2316 2335 SEQ ID NO: 6547 ttaatctttcataagtcaa 5201 5220 1 3 SEQ ID NO: 6002 tttttcccagacagtgtca 2402 2421 SEQ ID NO: 6548 agcttgtttgccagtgggc 6068 6087 1 3 SEQ ID NO: 6003 ttttcccagacagtgtcca 2405 2424 SEQ ID NO: 6549 tgaaatcattgaaaattta 2551 2570 1 3 SEQ ID NO: 6004 cattcagaaccagggaaga 2461 2480 SEQ ID NO: 6550 caggaggctttaagttgtc 7344 7363 1 3 SEQ ID NO: 6005 acaaactgtttatagtccc 2492 2511 SEQ ID NO: 6551 agcttgtttgccagtgggc 9202 9221 1 3 SEQ ID NO: 6006 ggatttcaatacatttcac 2547 2566 SEQ ID NO: 6552 taaagccattcaggtctct 5707 5723 1 3 SEQ ID NO: 6007 ttgtagaaatgaaagtaaa 2574 2593 SEQ ID NO: 6553 cttcatcgttcatttgagt 3015 3034 1 3 SEQ ID NO: 6008 ccaaaatttctctcggctt 2589 2809 SEQ ID NO: 6554 cttatgggatttcctaagg 3000 3019 1 3 SEQ ID NO: 6009 caaattttctctgctaaaa 2672 2691 SEQ ID NO: 6555 cttcatcgttcatttgagt 3637 3656 1 3 SEQ ID NO: 6010 tctgctggaaacaacaacg 2677 2696 SEQ ID NO: 6556 ttccatcacaaatcctttg 9355 9374 1 3 SEQ ID NO: 6011 cagaagctaagcaatgtcc 2720 2739 SEQ ID NO: 6557 tctcaaagagttacaacag 6848 6867 1 3 SEQ ID NO: 6012 taagattaaatatttactt 2721 2740 SEQ ID NO: 6558 tattgaaatatgatttttt 6847 6866 1 3 SEQ ID NO: 6013 aaagattactttgagaaat 2725 2744 SEQ ID NO: 6559 aattgttgaaagaaaaact 9160 9179 1 3 SEQ ID NO: 6014 aggaaatagttggattacc 2779 2498 SEQ ID NO: 6560 ggacaaggcccagaatctg 7443 7462 1 3 SEQ ID NO: 6015 attttgatgatgctggccc 2783 2602 SEQ ID NO: 6561 aaagaaacctatggcctta 9404 9423 1 3 SEQ ID NO: 6016 gaattatcttttaaacatt 2801 2826 SEQ ID NO: 6562 attcttaacgggattcctt 4192 4211 1 3 SEQ ID NO: 6017 ttaccaccagtttgaagtt 2687 2906 SEQ ID NO: 6563 taaagccattcaggtctct 3482 3501 1 3 SEQ ID NO: 6018 taagattaaatatttactt 2918 2937 SEQ ID NO: 6564 gacatgtttgaagggtgga 5242 5261 1 3 SEQ ID NO: 6019 aaagattactttgagaaat 2926 2945 SEQ ID NO: 6565 tgggctaaacgtttaattg 6261 6280 1 3 SEQ ID NO: 6020 aggaaatagttggattacc 2933 2952 SEQ ID NO: 6566 tattgaaatatgatttttt 8959 8978 1 3 SEQ ID NO: 6021 attttgatgatgctggccc 3006 3027 SEQ ID NO: 6567 agcttgtttgccagtgggc 8130 8149 1 3 SEQ ID NO: 6022 aggtttaaatggataattt 3010 3029 SEQ ID NO: 6568 aaagaaacctatggcctta 8267 8286 1 3 SEQ ID NO: 6023 cagaagctaagcaatgtcc 3032 3051 SEQ ID NO: 6569 attcttaacgggattcctt 7171 7190 1 3 SEQ ID NO: 6024 taagattaaatatttactt 3033 3052 SEQ ID NO: 6570 taaagccattcaggtctct 3945 3964 1 3 SEQ ID NO: 6025 aaagattactttgagaaat 3106 3125 SEQ ID NO: 6571 gacatgtttgaagggtgga 9348 9367 1 3 SEQ ID NO: 6026 aggaaatagttggattacc 3139 3158 SEQ ID NO: 6572 tgggctaaacgtttaattg 8354 8973 1 3 SEQ ID NO: 6027 attttgatgatgctggccc 3145 3164 SEQ ID NO: 6573 tattgaaatatgatttttt 7552 7571 1 3 SEQ ID NO: 6028 gaattatcttttaaacatt 353 3172 SEQ ID NO: 6574 agcttgtttgccagtgggc 5927 5946 1 3 SEQ ID NO: 6029 ttaccaccagtttgaagtt 3241 3260 SEQ ID NO: 6575 ggacaaggcccagaatctg 3349 3368 1 3 SEQ ID NO: 6030 ttgcagtgtatcggacaga 3262 3281 SEQ ID NO: 6576 aaagaaacctatggcctta 7717 7736 1 3 SEQ ID NO: 6031 cattcagaaccagggaaga 3299 3318 SEQ ID NO: 6577 attcttaacgggattcctt 8083 8102 1 3 SEQ ID NO: 6032 acaaactgtttatagtccc 3300 3319 SEQ ID NO: 6578 taaagccattcaggtctct 5792 5811 1 3 SEQ ID NO: 6033 ttaccaccagtttgaagtt 3321 3340 SEQ ID NO: 6579 aattgttgaaagaaaaact 4020 4039 1 3 SEQ ID NO: 6034 taagattaaatatttactt 3322 3341 SEQ ID NO: 6580 ggacaaggcccagaatctg 4019 4038 1 3 SEQ ID NO: 6035 aaagattactttgagaaat 3323 3342 SEQ ID NO: 6581 aaagaaacctatggcctta 6913 5932 1 3 SEQ ID NO: 6036 aggaaatagttggattacc 3343 3362 SEQ ID NO: 6582 attcttaacgggattcctt 7770 7789 1 3 SEQ ID NO: 6037 attttgatgatgctggccc 3346 3365 SEQ ID NO: 6583 taaagccattcaggtctct 3962 3981 1 3 SEQ ID NO: 6038 aggtttaaatggataattt 3365 3385 SEQ ID NO: 6584 gacatgtttgaagggtgga 4018 4037 1 3 SEQ ID NO: 6039 cagaagctaagcaatgtcc 3439 3458 SEQ ID NO: 6585 tgggctaaacgtttaattg 7519 7538 1 3 SEQ ID NO: 6040 taagattaaatatttactt 3530 3549 SEQ ID NO: 6586 tattgaaatatgatttttt 6215 6234 1 3 SEQ ID NO: 6041 aaagattactttgagaaat 3540 3559 SEQ ID NO: 6587 agcttgtttgccagtgggc 6020 6039 1 3 SEQ ID NO: 6042 aggaaatagttggattacc 3671 3690 SEQ ID NO: 6588 tgaaatcattgaaaattta 5542 5561 1 3 SEQ ID NO: 6043 attttgatgatgctggccc 3685 3704 SEQ ID NO: 6589 caggaggctttaagttgtc 4840 4859 1 3 SEQ ID NO: 6044 gaattatcttttaaacatt 3698 3717 SEQ ID NO: 6590 agcttgtttgccagtgggc 6818 6637 1 3 SEQ ID NO: 6045 ttaccaccagtttgaagtt 3762 3761 SEQ ID NO: 6591 taaagccattcaggtctct 3931 3950 1 3 SEQ ID NO: 6046 ttgcagtgtatcggacaga 3802 3821 SEQ ID NO: 6592 aattgttgaaagaaaaact 9111 9130 1 3 SEQ ID NO: 6047 cattcagaaccagggaaga 3904 3923 SEQ ID NO: 6593 taaagccattcaggtctct 5680 5699 1 3 SEQ ID NO: 6048 acaaactgtttatagtccc 3926 3945 SEQ ID NO: 6594 ttaatctttcataagtcaa 5813 5632 1 3 SEQ ID NO: 6049 ggatttcaatacatttcac 3996 4015 SEQ ID NO: 6595 ggacaaggcccagaatctg 8940 8959 1 3 SEQ ID NO: 6050 ttgtagaaatgaaagtaaa 4013 4032 SEQ ID NO: 6596 attcttaacgggattcctt 5385 5408 1 3 SEQ ID NO: 6051 ctgaacagtgagctgcagt 4028 4047 SEQ ID NO: 6597 aattgttgaaagaaaaact 5815 5834 1 3 SEQ ID NO: 6052 aatccaatctcctctttgc 4068 4067 SEQ ID NO: 6598 agcttgtttgccagtgggc 5937 5956 1 3 SEQ ID NO: 6053 atttgattttcaagcaaag 4140 4159 SEQ ID NO: 6599 ttaatctttcataagtcaa 5163 5182 1 3 SEQ ID NO: 6054 ttttgattttcaagcaaat 4141 4160 SEQ ID NO: 6600 agcttgtttgccagtgggc 5162 5181 1 3 SEQ ID NO: 6055 tgatttcaagcaaatgcag 4191 4210 SEQ ID NO: 6601 tgaaatcattgaaaattta 6167 5186 1 3 SEQ ID NO: 6056 atgctgtttgggaaattgg 4209 4228 SEQ ID NO: 6602 caggaggctttaagttgtc 5059 6076 1 3 SEQ ID NO: 6057 tgctgttttttggaaatgc 4236 4255 SEQ ID NO: 6603 agcttgtttgccagtgggc 7839 7858 1 3 SEQ ID NO: 6058 aaaaaaatacactggacgt 4352 4371 SEQ ID NO: 6604 taaagccattcaggtctct 7637 7556 1 3 SEQ ID NO: 6059 actggagcttagtaagggc 4384 4403 SEQ ID NO: 6605 cttcatcgttcatttgagt 7619 7538 1 3 SEQ ID NO: 6060 cttctggaaagagggtcat 4453 4472 SEQ ID NO: 6606 cttatgggatttcctaagg 6845 6864 1 3 SEQ ID NO: 6061 ggaaaagggtcatggaaat 4477 4496 SEQ ID NO: 6607 cttcatcgttcatttgagt 9257 9276 1 3 SEQ ID NO: 6062 gggcctgccccagatttct 4482 4501 SEQ ID NO: 6608 ttccatcacaaatcctttg 7527 7546 1 3 SEQ ID NO: 6063 ttctcagataggggaacac 4483 4502 SEQ ID NO: 6609 tctcaaagagttacaacag 7526 7545 1 3 SEQ ID NO: 6064 gatgaaagaattaagggga 4586 4587 SEQ ID NO: 6610 tattgaaatatgatttttt 7414 7433 1 3 SEQ ID NO: 6065 cttggactgtcaaataagt 4612 4631 SEQ ID NO: 6611 aattgttgaaagaaaaact 6217 6290 1 3 SEQ ID NO: 6066 ggaaaagggtcatggaaat 4616 4635 SEQ ID NO: 6612 ggacaaggcccagaatctg 6968 8957 1 3 SEQ ID NO: 6067 gggcctgccccagatttct 4668 4687 SEQ ID NO: 6613 aaagaaacctatggcctta 7184 7203 1 3 SEQ ID NO: 6068 cttggactgtcaaataagt 4684 4703 SEQ ID NO: 6614 attcttaacgggattcctt 8213 8232 1 3 SEQ ID NO: 6069 ggatttcaatacatttcac 4724 4743 SEQ ID NO: 6615 taaagccattcaggtctct 8947 8966 1 3 SEQ ID NO: 6070 ttgtagaaatgaaagtaaa 4736 4757 SEQ ID NO: 6616 gacatgtttgaagggtgga 6301 6320 1 3 SEQ ID NO: 6071 ccaaaatttctctcggctt 4760 4779 SEQ ID NO: 6617 tgggctaaacgtttaattg 1891 1910 1 3 SEQ ID NO: 6072 caaattttctctgctaaaa 4805 4825 SEQ ID NO: 6618 tattgaaatatgatttttt 9657 8676 1 3 SEQ ID NO: 6073 tctgctggaaacaacaacg 4831 4850 SEQ ID NO: 6619 agcttgtttgccagtgggc 7622 7641 1 3 SEQ ID NO: 6074 cagaagctaagcaatgtcc 4855 4874 SEQ ID NO: 6620 tgaaatcattgaaaattta 6546 6565 1 3 SEQ ID NO: 6075 taagattaaatatttactt 4906 4925 SEQ ID NO: 6621 tgaaatcattgaaaattta 8009 8028 1 3 SEQ ID NO: 6076 aaagattactttgagaaat 4907 4926 SEQ ID NO: 6622 aaagaaacctatggcctta 6008 8027 1 3 SEQ ID NO: 6077 aggaaatagttggattacc 4918 4937 SEQ ID NO: 6623 attcttaacgggattcctt 5849 588 1 3 SEQ ID NO: 6078 attttgatgatgctggccc 5050 5069 SEQ ID NO: 6624 taaagccattcaggtctct 3164 3183 1 3 SEQ ID NO: 6079 gaattatcttttaaacatt 5101 5120 SEQ ID NO: 6625 gacatgtttgaagggtgga 8002 8021 1 3 SEQ ID NO: 6080 ttaccaccagtttgaagtt 5130 5149 SEQ ID NO: 6626 tgggctaaacgtttaattg 8065 8085 1 3 SEQ ID NO: 6081 taagattaaatatttactt 5147 5165 SEQ ID NO: 6627 tattgaaatatgatttttt 5892 5911 1 3 SEQ ID NO: 6082 aaagattactttgagaaat 5175 5194 SEQ ID NO: 6628 agcttgtttgccagtgggc 5933 5952 1 3 SEQ ID NO: 6083 aggaaatagttggattacc 5214 5233 SEQ ID NO: 6629 tgaaatcattgaaaattta 6832 6851 1 3 SEQ ID NO: 6084 attttgatgatgctggccc 5268 5287 SEQ ID NO: 6630 caggaggctttaagttgtc 9072 9091 1 3 SEQ ID NO: 6085 gaattatcttttaaacatt 5336 5355 SEQ ID NO: 6631 agcttgtttgccagtgggc 6956 6974 1 3 SEQ ID NO: 6086 ttaccaccagtttgaagtt 5364 5383 SEQ ID NO: 6632 taaagccattcaggtctct 9401 9420 1 3 SEQ ID NO: 6087 ctgaaaaagttagtgaaag 5557 5576 SEQ ID NO: 6633 aattgttgaaagaaaaact 8820 8839 1 3 SEQ ID NO: 6088 tttttcccagacagtgtca 5635 5654 SEQ ID NO: 6634 taaagccattcaggtctct 7702 7721 1 3 SEQ ID NO: 6089 ttttcccagacagtgtcca 5728 5747 SEQ ID NO: 6635 ttaatctttcataagtcaa 9565 9564 1 3 SEQ ID NO: 6090 cattcagaaccagggaaga 5751 5770 SEQ ID NO: 6636 ggacaaggcccagaatctg 5771 5790 1 3 SEQ ID NO: 6091 acaaactgtttatagtccc 5762 5781 SEQ ID NO: 6637 attcttaacgggattcctt 9378 9397 1 3 SEQ ID NO: 6092 ggatttcaatacatttcac 5777 5796 SEQ ID NO: 6638 aattgttgaaagaaaaact 6564 6583 1 3 SEQ ID NO: 6093 ttgtagaaatgaaagtaaa 5818 5937 SEQ ID NO: 6639 agcttgtttgccagtgggc 6832 6851 1 3 SEQ ID NO: 6094 ccaaaatttctctcggctt 5829 5848 SEQ ID NO: 6640 ggacaaggcccagaatctg 8453 8472 1 3 SEQ ID NO: 6095 caaattttctctgctaaaa 5845 5864 SEQ ID NO: 6641 aaagaaacctatggcctta 7727 7746 1 3 SEQ ID NO: 6096 tctgctggaaacaacaacg 5853 5872 SEQ ID NO: 6642 attcttaacgggattcctt 7111 7130 1 3 SEQ ID NO: 6097 cagaagctaagcaatgtcc 5909 5928 SEQ ID NO: 6643 taaagccattcaggtctct 5794 5813 1 3 SEQ ID NO: 6098 taagattaaatatttactt 5922 5941 SEQ ID NO: 6644 gacatgtttgaagggtgga 8063 8082 1 3 SEQ ID NO: 6099 aaagattactttgagaaat 5923 5942 SEQ ID NO: 6645 tgggctaaacgtttaattg 8062 8081 1 3 SEQ ID NO: 6100 aggaaatagttggattacc 5950 5969 SEQ ID NO: 6646 tattgaaatatgatttttt 7596 7615 1 3 SEQ ID NO: 6101 attttgatgatgctggccc 6060 8079 SEQ ID NO: 6647 agcttgtttgccagtgggc 4019 4038 1 3 SEQ ID NO: 6102 gaattatcttttaaacatt 6095 5114 SEQ ID NO: 6648 tgaaatcattgaaaattta 5664 5583 1 3 SEQ ID NO: 6103 ttaccaccagtttgaagtt 6146 6165 SEQ ID NO: 6649 tgaaatcattgaaaattta 7428 7447 1 3 SEQ ID NO: 6104 taagattaaatatttactt 6237 6256 SEQ ID NO: 6650 aaagaaacctatggcctta 6399 6418 1 3 SEQ ID NO: 6105 aaagattactttgagaaat 6331 6350 SEQ ID NO: 6651 attcttaacgggattcctt 7969 7988 1 3 SEQ ID NO: 6106 aggaaatagttggattacc 6442 6461 SEQ ID NO: 6652 taaagccattcaggtctct 9287 9306 1 3 SEQ ID NO: 6107 attttgatgatgctggccc 6506 6525 SEQ ID NO: 6653 gacatgtttgaagggtgga 7257 7276 1 3 SEQ ID NO: 6108 aggtttaaatggataattt 6544 6563 SEQ ID NO: 6654 tgggctaaacgtttaattg 7860 7679 1 3 SEQ ID NO: 6109 ggatttcaatacatttcac 6675 6694 SEQ ID NO: 6655 tattgaaatatgatttttt 8803 8622 1 3 SEQ ID NO: 6110 ttgtagaaatgaaagtaaa 6677 6696 SEQ ID NO: 6656 agcttgtttgccagtgggc 3164 3183 1 3 SEQ ID NO: 6111 ctgaacagtgagctgcagt 6693 6712 SEQ ID NO: 6657 tgaaatcattgaaaattta 7593 7612 1 3 SEQ ID NO: 6112 aatccaatctcctctttgc 6708 6727 SEQ ID NO: 6658 caggaggctttaagttgtc 8163 8172 1 3 SEQ ID NO: 6113 atttgattttcaagcaaag 6709 6726 SEQ ID NO: 6659 agcttgtttgccagtgggc 8152 7171 1 3 SEQ ID NO: 6114 ttttgattttcaagcaaat 6872 6891 SEQ ID NO: 6660 taaagccattcaggtctct 8276 7297 1 3 SEQ ID NO: 6115 acaaactgtttatagtccc 6911 6930 SEQ ID NO: 6661 aattgttgaaagaaaaact 5782 5801 1 3 SEQ ID NO: 6116 ggatttcaatacatttcac 6972 6991 SEQ ID NO: 6662 taaagccattcaggtctct 7811 7830 1 3 SEQ ID NO: 6117 ttgtagaaatgaaagtaaa 7060 7079 SEQ ID NO: 6663 ttaatctttcataagtcaa 8887 8906 1 3 SEQ ID NO: 6118 ttaccaccagtttgaagtt 7061 7080 SEQ ID NO: 6664 ggacaaggcccagaatctg 8885 8905 1 3 SEQ ID NO: 6119 ttgcagtgtatcggacaga 7112 7131 SEQ ID NO: 6665 attcttaacgggattcctt 8457 8476 1 3 SEQ ID NO: 6120 cattcagaaccagggaaga 7113 7132 SEQ ID NO: 6666 aattgttgaaagaaaaact 7556 7575 1 3 SEQ ID NO: 6121 acaaactgtttatagtccc 7149 7168 SEQ ID NO: 6667 agcttgtttgccagtgggc 8030 8049 1 3 SEQ ID NO: 6122 ggatttcaatacatttcac 7164 7183 SEQ ID NO: 6668 ttaatctttcataagtcaa 8730 8749 1 3 SEQ ID NO: 6123 ttgtagaaatgaaagtaaa 7580 7599 SEQ ID NO: 6669 agcttgtttgccagtgggc 9305 9324 1 3 SEQ ID NO: 6124 ccaaaatttctctcggctt 7594 7613 SEQ ID NO: 6670 tgaaatcattgaaaattta 9197 9216 1 3 SEQ ID NO: 6125 caaattttctctgctaaaa 7601 7620 SEQ ID NO: 6671 caggaggctttaagttgtc 7716 7735 1 3 SEQ ID NO: 6126 tctgctggaaacaacaacg 7704 7723 SEQ ID NO: 6672 agcttgtttgccagtgggc 6123 6142 1 3 SEQ ID NO: 6127 cagaagctaagcaatgtcc 7757 7776 SEQ ID NO: 6673 taaagccattcaggtctct 8031 8050 1 3 SEQ ID NO: 6128 taagattaaatatttactt 7789 7808 SEQ ID NO: 6674 cttcatcgttcatttgagt 7936 7955 1 3 SEQ ID NO: 6129 aaagattactttgagaaat 7807 7826 SEQ ID NO: 6675 cttatgggatttcctaagg 9116 9135 1 3 SEQ ID NO: 6130 aggaaatagttggattacc 8032 8051 SEQ ID NO: 6676 cttcatcgttcatttgagt 7756 7775 1 3 SEQ ID NO: 6131 attttgatgatgctggccc 8110 8129 SEQ ID NO: 6677 ttccatcacaaatcctttg 9069 9088 1 3 SEQ ID NO: 6132 gaattatcttttaaacatt 8365 8384 SEQ ID NO: 6678 tctcaaagagttacaacag 9551 9570 1 3 SEQ ID NO: 6133 ttaccaccagtttgaagtt 8900 8919 SEQ ID NO: 6679 tattgaaatatgatttttt 7134 7153 1 3 SEQ ID NO: 6134 taagattaaatatttactt 9303 9322 SEQ ID NO: 6680 aattgttgaaagaaaaact 3688 3707 1 3

Start End Match Start End Match Source Index Index Index Index # SEQ ID tttttttttttttttttttt 9446 9465 SEQ ID tttttttttttttttttttt 9466 9485 2 NO: 4088 NO: 4661 SEQ ID tttttttttttttttttttt 9446 9465 SEQ ID tttttttttttttttttttt 9465 9484 1 NO: 4347 NO: 5229 SEQ ID tttttttttttttttttttt 9447 9466 SEQ ID tttttttttttttttttttt 9466 9485 1 NO: 4348 NO: 5230

TABLE 15 Sequences of Exemplary Gene Targets gi|4502152|ref|Nm_000384.1| Homo sapiens apolipoprotein B (including Ag(x) antigen) (APOB), mRNA ATTCCCACCGGGACCTGCGGGGCTGAGTGCCCTTCTCGGTTGCTGCCGCTGAGGAGCCCGCCCAGCCAGC CAGGGCCGCGAGGCCGAGGCCAGGCCGCAGCCCAGGAGCCGCCCCACCGCAGCTGGCGATGGACCCGCCG AGGCCCGCGCTGCTGGCGCTGCTGGCGCTGCCTGCGCTGCTGCTGCTGCTGCTGGCGGGCGCCAGGGCCG AAGAGGAAATGCTGGAAAATGTCAGCCTGGTCTGTCCAAAAGATGCGACCCGATTCAAGCACCTCCGGAA GTACACATACAACTATGAGGCTGAGAGTTCCAGTGGAGTCCCTGGGACTGCTGATTCAAGAAGTGCCACC AGGATCAACTGCAAGGTTGAGCTGGAGGTTCCCCAGCTCTGCAGCTTCATCCTGAAGACCAGCCAGTGCA CCCTGAAAGAGGTGTATGGCTTCAACCCTGAGGGCAAAGCCTTGCTGAAGAAAACCAAGAACTCTGAGGA GTTTGCTGCAGCCATGTCCAGGTATGAGCTCAAGCTGGCCATTCCAGAAGGGAAGCAGGTTTTCCTTTAC CCGGAGAAAGATGAACCTACTTACATCCTGAACATCAAGAGGGGCATCATTTCTGCCCTCCTGGTTCCCC CAGAGACAGAAGAAGCCAAGCAAGTGTTGTTTCTGGATACCGTGTATGGAAACTGCTCCACTCACTTTAC CGTCAAGACGAGGAAGGGCAATGTGGCAACAGAAATATCCACTGAAAGAGACCTGGGGCAGTGTGATCGC TTCAAGCCCATCCGCACAGGCATCAGCCCACTTGCTCTCATCAAAGGCATGACCCGCCCCTTGTCAACTC TGATCAGCAGCAGCCAGTCCTGTCAGTACACACTGGACGCTAAGAGGAAGCATGTGGCAGAAGCCATCTG CAAGGAGCAACACCTCTTCCTGCCTTTCTCCTACAACAATAAGTATGGGATGGTAGCACAAGTGACACAG ACTTTGAAACTTGAAGACACACCAAAGATCAACAGCCGCTTCTTTGGTGAAGGTACTAAGAAGATGGGCC TCGCATTTGAGAGCACCAAATCCACATCACCTCCAAAGCAGGCCGAAGCTGTTTTGAAGACTCTCCAGGA ACTGAAAAAACTAACCATCTCTGAGCAAAATATCCAGAGAGCTAATCTCTTCAATAAGCTGGTTACTGAG CTGAGAGGCCTCAGTGATGAAGCAGTCACATCTCTCTTGCCACAGCTGATTGAGGTGTCCAGCCCCATCA CTTTACAAGCCTTGGTTCAGTGTGGACAGCCTCAGTGCTCCACTCACATCCTCCAGTGGCTGAAACGTGT GCATGCCAACCCCCTTCTGATAGATGTGGTCACCTACCTGGTGGCCCTGATCCCCGAGCCCTCAGCACAG CAGCTGCGAGAGATCTTCAACATGGCGAGGGATCAGCGCAGCCGAGCCACCTTGTATGCGCTGAGCCACG CGGTCAACAACTATCATAAGACAAACCCTACAGGGACCCAGGAGCTGCTGGACATTGCTAATTACCTGAT GGAACAGATTCAAGATGACTGCACTGGGGATGAAGATTACACCTATTTGATTCTGCGGGTCATTGGAAAT ATGGGCCAAACCATGGAGCAGTTAACTCCAGAACTCAAGTCTTCAATCCTCAAATGTGTCCAAAGTACAA AGCCATCACTGATGATCCAGAAAGCTGCCATCCAGGCTCTGCGGAAAATGGAGCCTAAAGACAAGGACCA GGAGGTTCTTCTTCAGACTTTCCTTGATGATGCTTCTCCGGGAGATAAGCGACTGGCTGCCTATCTTATG TTGATGAGGAGTCCTTCACAGGCAGATATTAACAAAATTGTCCAAATTCTACCATGGGAACAGAATGAGC AAGTGAAGAACTTTGTGGCTTCCCATATTGCCAATATCTTGAACTCAGAAGAATTGGATATCCAAGATCT GAAAAAGTTAGTGAAAGAAGCTCTGAAAGAATCTCAACTTCCAACTGTCATGGACTTCAGAAAATTCTCT CGGAACTATCAACTCTACAAATCTGTTTCTCTTCCATCACTTGACCCAGCCTCAGCCAAAATAGAAGGGA ATCTTATATTTGATCCAAATAACTACCTTCCTAAAGAAAGCATGCTGAAAACTACCCTCACTGCCTTTGG ATTTGCTTCAGCTGACCTCATCGAGATTGGCTTGGAAGGAAAAGGCTTTGAGCCAACATTGGAAGCTCTT TTTGGGAAGCAAGGATTTTTCCCAGACAGTGTCAACAAAGCTTTGTACTGGGTTAATGGTCAAGTTCCTG ATGGTGTCTCTAAGGTCTTAGTGGACCACTTTGGCTATACCAAAGATGATAAACATGAGCAGGATATGGT AAATGGAATAATGCTCAGTGTTGAGAAGCTGATTAAAGATTTGAAATCCAAAGAAGTCCCGGAAGCCAGA GCCTACCTCCGCATCTTGGGAGAGGAGCTTGGTTTTGCCAGTCTCCATGACCTCCAGCTCCTGGGAAAGC TGCTTCTGATGGGTGCCCGCACTCTGCAGGGGATCCCCCAGATGATTGGAGAGGTCATCAGGAAGGGCTC AAAGAATGACTTTTTTCTTCACTACATCTTCATGGAGAATGCCTTTGAACTCCCCACTGGAGCTGGATTA CAGTTGCAAATATCTTCATCTGGAGTCATTGCTCCCGGAGCCAAGGCTGGAGTAAAACTGGAAGTAGCCA ACATGCAGGCTGAACTGGTGGCAAAACCCTCCGTGTCTGTGGAGTTTGTGACAAATATGGGCATCATCAT TCCGGACTTCGCTAGGAGTGGGGTCCAGATGAACACCAACTTCTTCCACGAGTCGGGTCTGGAGGCTCAT GTTGCCCTAAAAGCTGGGAAGCTGAAGTTTATCATTCCTTCCCCAAAGAGACCAGTCAAGCTGCTCAGTG GAGGCAACACATTACATTTGGTCTCTACCACCAAAACGGAGGTGATCCCACCTCTCATTGAGAACAGGCA GTCCTGGTCAGTTTGCAAGCAAGTCTTTCCTGGCCTGAATTACTGCACCTCAGGCGCTTACTCCAACGCC AGCTCCACAGACTCCGCCTCCTACTATCCGCTGACCGGGGACACCAGATTAGAGCTGGAACTGAGGCCTA CAGGAGAGATTGAGCAGTATTCTGTCAGCGCAACCTATGAGCTCCAGAGAGAGGACAGAGCCTTGGTGGA TACCCTGAAGTTTGTAACTCAAGCAGAAGGTGCGAAGCAGACTGAGGCTACCATGACATTCAAATATAAT CGGCAGAGTATGACCTTGTCCAGTGAAGTCCAAATTCCGGATTTTGATGTTGACCTCGGAACAATCCTCA GAGTTAATGATGAATCTACTGAGGGCAAAACGTCTTACAGACTCACCCTGGACATTCAGAACAAGAAAAT TACTGAGGTCGCCCTCATGGGCCACCTAAGTTGTGACACAAAGGAAGAAAGAAAAATCAAGGGTGTTATT TCCATACCCCGTTTGCAAGCAGAAGCCAGAAGTGAGATCCTCGCCCACTGGTCGCCTGCCAAACTGCTTC TCCAAATGGACTCATCTGCTACAGCTTATGGCTCCACAGTTTCCAAGAGGGTGGCATGGCATTATGATGA AGAGAAGATTGAATTTGAATGGAACACAGGCACCAATGTAGATACCAAAAAAATGACTTCCAATTTCCCT GTGGATCTCTCCGATTATCCTAAGAGCTTGCATATGTATGCTAATAGACTCCTGGATCACAGAGTCCCTG AAACAGACATGACTTTCCGGCACGTGGGTTCCAAATTAATAGTTGCAATGAGCTCATGGCTTCAGAAGGC ATCTGGGAGTCTTCCTTATACCCAGACTTTGCAAGACCACCTCAATAGCCTGAAGGAGTTCAACCTCCAG AACATGGGATTGCCAGACTTCCACATCCCAGAAAACCTCTTCTTAAAAAGCGATGGCCGGGTCAAATATA CCTTGAACAAGAACAGTTTGAAAATTGAGATTCCTTTGCCTTTTGGTGGCAAATCCTCCAGAGATCTAAA GATGTTAGAGACTGTTAGGACACCAGCCCTCCACTTCAAGTCTGTGGGATTCCATCTGCCATCTCGAGAG TTCCAAGTCCCTACTTTTACCATTCCCAAGTTGTATCAACTGCAAGTGCCTCTCCTGGGTGTTCTAGACC TCTCCACGAATGTCTACAGCAACTTGTACAACTGGTCCGCCTCCTACAGTGGTGGCAACACCAGCACAGA CCATTTCAGCCTTCGGGCTCGTTACCACATGAAGGCTGACTCTGTGGTTGACCTGCTTTCCTACAATGTG CAAGGATCTGGAGAAACAACATATGACCACAAGAATACGTTCACACTATCATGTGATGGGTCTCTACGCC ACAAATTTCTAGATTCGAATATCAAATTCAGTCATGTAGAAAAACTTGGAAACAACCCAGTCTCAAAAGG TTTACTAATATTCGATGCATCTAGTTCCTGGGGACCACAGATGTCTGCTTCAGTTCATTTGGACTCCAAA AAGAAACAGCATTTGTTTGTCAAAGAAGTCAAGATTGATGGGCAGTTCAGAGTCTCTTCGTTCTATGCTA AAGGCACATATGGCCTGTCTTGTCAGAGGGATCCTAACACTGGCCGGCTCAATGGAGAGTCCAACCTGAG GTTTAACTCCTCCTACCTCCAAGGCACCAACCAGATAACAGGAAGATATGAAGATGGAACCCTCTCCCTC ACCTCCACCTCTGATCTGCAAAGTGGCATCATTAAAAATACTGCTTCCCTAAAGTATGAGAACTACGAGC TGACTTTAAAATCTGACACCAATGGGAAGTATAAGAACTTTGCCACTTCTAACAAGATGGATATGACCTT CTCTAAGCAAAATGCACTGCTGCGTTCTGAATATCAGGCTGATTACGAGTCATTGAGGTTCTTCAGCCTG CTTTCTGGATCACTAAATTCCCATGGTCTTGAGTTAAATGCTGACATCTTAGGCACTGACAAAATTAATA GTGGTGCTCACAAGGCGACACTAAGGATTGGCCAAGATGGAATATCTACCAGTGCAACGACCAACTTGAA GTGTAGTCTCCTGGTGCTGGAGAATGAGCTGAATGCAGAGCTTGGCCTCTCTGGGGCATCTATGAAATTA ACAACAAATGGCCGCTTCAGGGAACACAATGCAAAATTCAGTCTGGATGGGAAAGCCGCCCTCACAGAGC TATCACTGGGAAGTGCTTATCAGGCCATGATTCTGGGTGTCGACAGCAAAAACATTTTCAACTTCAAGGT CAGTCAAGAAGGACTTAAGCTCTCAAATGACATGATGGGCTCATATGCTGAAATGAAATTTGACCACACA AACAGTCTGAACATTGCAGGCTTATCACTGGACTTCTCTTCAAAACTTGACAACATTTACAGCTCTGACA AGTTTTATAAGCAAACTGTTAATTTACAGCTACAGCCCTATTCTCTGGTAACTACTTTAAACAGTGACCT GAAATACAATGCTCTGGATCTCACCAACAATGGGAAACTACGGCTAGAACCCCTGAAGCTGCATGTGGCT GGTAACCTAAAAGGAGCCTACCAAAATAATGAAATAAAACACATCTATGCCATCTCTTCTGCTGCCTTAT CAGCAAGCTATAAAGCAGACACTGTTGCTAAGGTTCAGGGTGTGGAGTTTAGCCATCGGCTCAACACAGA CATCGCTGGGCTGGCTTCAGCCATTGACATGAGCACAAACTATAATTCAGACTCACTGCATTTCAGCAAT GTCTTCCGTTCTGTAATGGCCCCGTTTACCATGACCATCGATGCACATACAAATGGCAATGGGAAACTCG CTCTCTGGGGAGAACATACTGGGCAGCTGTATAGCAAATTCCTGTTGAAAGCAGAACCTCTGGCATTTAC TTTCTCTCATGATTACAAAGGCTCCACAAGTCATCATCTCGTGTCTAGGAAAAGCATCAGTGCAGCTCTT GAACACAAAGTCAGTGCCCTGCTTACTCCAGCTGAGCAGACAGGCACCTGGAAACTCAAGACCCAATTTA ACAACAATGAATACAGCCAGGACTTGGATGCTTACAACACTAAAGATAAAATTGGCGTGGAGCTTACTGG ACGAACTCTGGCTGACCTAACTCTACTAGACTCCCCAATTAAAGTGCCACTTTTACTCAGTGAGCCCATC AATATCATTGATGCTTTAGAGATGAGAGATGCCGTTGAGAAGCCCCAAGAATTTACAATTGTTGCTTTTG TAAAGTATGATAAAAACCAAGATGTTCACTCCATTAACCTCCCATTTTTTGAGACCTTGCAAGAATATTT TGAGAGGAATCGACAAACCATTATAGTTGTAGTGGAAAACGTACAGAGAAACCTGAAGCACATCAATATT GATCAATTTGTAAGAAAATACAGAGCAGCCCTGGGAAAACTCCCACAGCAAGCTAATGATTATCTGAATT CATTCAATTGGGAGAGACAAGTTTCACATGCCAAGGAGAAACTGACTGCTCTCACAAAAAAGTATAGAAT TACAGAAAATGATATACAAATTGCATTAGATGATGCCAAAATCAACTTTAATGAAAAACTATCTCAACTG CAGACATATATGATACAATTTGATCAGTATATTAAAGATAGTTATGATTTACATGATTTGAAAATAGCTA TTGCTAATATTATTGATGAAATCATTGAAAAATTAAAAAGTCTTGATGAGCACTATCATATCCGTGTAAA TTTAGTAAAAACAATCCATGATCTACATTTGTTTATTGAAAATATTGATTTTAACAAAAGTGGAAGTAGT ACTGCATCCTGGATTCAAAATGTGGATACTAAGTACCAAATCAGAATCCAGATACAAGAAAAACTGCAGC AGCTTAAGAGACACATACAGAATATAGACATCCAGCACCTAGCTGGAAAGTTAAAACAACACATTGAGGC TATTGATGTTAGAGTGCTTTTAGATCAATTGGGAACTACAATTTCATTTGAAAGAATAAATGATGTTCTT GAGCATGTCAAACACTTTGTTATAAATCTTATTGGGGATTTTGAAGTAGCTGAGAAAATCAATGCCTTCA GAGCCAAAGTCCATGAGTTAATCGAGAGGTATGAAGTAGACCAACAAATCCAGGTTTTAATGGATAAATT AGTAGAGTTGACCCACCAATACAAGTTGAAGGAGACTATTCAGAAGCTAAGCAATGTCCTACAACAAGTT AAGATAAAAGATTACTTTGAGAAATTGGTTGGATTTATTGATGATGCTGTGAAGAAGCTTAATGAATTAT CTTTTAAAACATTCATTGAAGATGTTAACAAATTCCTTGACATGTTGATAAAGAAATTAAAGTCATTTGA TTACCACCAGTTTGTAGATGAAACCAATGACAAAATCCGTGAGGTGACTCAGAGACTCAATGGTGAAATT CAGGCTCTGGAACTACCACAAAAAGCTGAAGCATTAAAACTGTTTTTAGAGGAAACCAAGGCCACAGTTG CAGTGTATCTGGAAAGCCTACAGGACACCAAAATAACCTTAATCATCAATTGGTTACAGGAGGCTTTAAG TTCAGCATCTTTGGCTCACATGAAGGCCAAATTCCGAGAGACTCTAGAAGATACACGAGACCGAATGTAT CAAATGGACATTCAGCAGGAACTTCAACGATACCTGTCTCTGGTAGGCCAGGTTTATAGCACACTTGTCA CCTACATTTCTGATTGGTGGACTCTTGCTGCTAAGAACCTTACTGACTTTGCAGAGCAATATTCTATCCA AGATTGGGCTAAACGTATGAAAGCATTGGTAGAGCAAGGGTTCACTGTTCCTGAAATCAAGACCATCCTT GGGACCATGCCTGCCTTTGAAGTCAGTCTTCAGGCTCTTCAGAAAGCTACCTTCCAGACACCTGATTTTA TAGTCCCCCTAACAGATTTGAGGATTCCATCAGTTCAGATAAACTTCAAAGACTTAAAAAATATAAAAAT CCCATCCAGGTTTTCCACACCAGAATTTACCATCCTTAACACCTTCCACATTCCTTCCTTTACAATTGAC TTTGTCGAAATGAAAGTAAAGATCATCAGAACCATTGACCAGATGCAGAACAGTGAGCTGCAGTGGCCCG TTCCAGATATATATCTCAGGGATCTGAAGGTGGAGGACATTCCTCTAGCGAGAATCACCCTGCCAGACTT CCGTTTACCAGAAATCGCAATTCCAGAATTCATAATCCCAACTCTCAACCTTAATGATTTTCAAGTTCCT GACCTTCACATACCAGAATTCCAGCTTCCCCACATCTCACACACAATTGAAGTACCTACTTTTGGCAAGC TATACAGTATTCTGAAAATCCAATCTCCTCTTTTCACATTAGATGCAAATGCTGACATAGGGAATGGAAC CACCTCAGCAAACGAAGCAGGTATCGCAGCTTCCATCACTGCCAAAGGAGAGTCCAAATTAGAAGTTCTC AATTTTGATTTTCAAGCAAATGCACAACTCTCAAACCCTAAGATTAATCCGCTGGCTCTGAAGGAGTCAG TGAAGTTCTCCAGCAAGTACCTGAGAACGGAGCATGGGAGTGAAATGCTGTTTTTTGGAAATGCTATTGA GGGAAAATCAAACACAGTGGCAAGTTTACACACAGAAAAAAATACACTGGAGCTTAGTAATGGAGTGATT GTCAAGATAAACAATCAGCTTACCCTGGATAGCAACACTAAATACTTCCACAAATTGAACATCCCCAAAC TGGACTTCTCTAGTCAGGCTGACCTGCGCAACGAGATCAAGACACTGTTGAAAGCTGGCCACATAGCATG GACTTCTTCTGGAAAAGGGTCATGGAAATGGGCCTGCCCCAGATTCTCAGATGAGGGAACACATGAATCA CAAATTAGTTTCACCATAGAAGGACCCCTCACTTCCTTTGGACTGTCCAATAAGATCAATAGCAAACACC TAAGAGTAAACCAAAACTTGGTTTATGAATCTGGCTCCCTCAACTTTTCTAAACTTGAAATTCAATCACA AGTCGATTCCCAGCATGTGGGCCACAGTGTTCTAACTGCTAAAGGCATGGCACTGTTTGGAGAAGGGAAG GCAGAGTTTACTGGGAGGCATGATGCTCATTTAAATGGAAAGGTTATTGGAACTTTGAAAAATTCTCTTT TCTTTTCAGCCCAGCCATTTGAGATCACGGCATCCACAAACAATGAAGGGAATTTGAAAGTTCGTTTTCC ATTAAGGTTAACAGGGAAGATAGACTTCCTGAATAACTATGCACTGTTTCTGAGTCCCAGTGCCCAGCAA GCAAGTTGGCAAGTAAGTGCTAGGTTCAATCAGTATAAGTACAACCAAAATTTCTCTGCTGGAAACAACG AGAACATTATGGAGGCCCATGTAGGAATAAATGGAGAAGCAAATCTGGATTTCTTAAACATTCCTTTAAC AATTCCTGAAATGCGTCTACCTTACACAATAATCACAACTCCTCCACTGAAAGATTTCTCTCTATGGGAA AAAACAGGCTTGAAGGAATTCTTGAAAACGACAAAGCAATCATTTGATTTAAGTGTAAAAGCTCAGTATA AGAAAAACAAACACAGGCATTCCATCACAAATCCTTTGGCTGTGCTTTGTGAGTTTATCAGTCAGAGCAT CAAATCCTTTGACAGGCATTTTGAAAAAAACAGAAACAATGCATTAGATTTTGTCACCAAATCCTATAAT GAAACAAAAATTAAGTTTGATAAGTACAAAGCTGAAAAATCTCACGACGAGCTCCCCAGGACCTTTCAAA TTCCTGGATACACTGTTCCAGTTGTCAATGTTGAAGTGTCTCCATTCACCATAGAGATGTCGGCATTCGG CTATGTGTTCCCAAAAGCAGTCAGCATGCCTAGTTTCTCCATCCTAGGTTCTGACGTCCGTGTGCCTTCA TACACATTAATCCTGCCATCATTAGAGCTGCCAGTCCTTCATGTCCCTAGAAATCTCAAGCTTTCTCTTC CACATTTCAAGGAATTGTGTACCATAAGCCATATTTTTATTCCTGCCATGGGCAATATTACCTATGATTT CTCCTTTAAATCAAGTGTCATCACACTGAATACCAATGCTGAACTTTTTAACCAGTCAGATATTGTTGCT CATCTCCTTTCTTCATCTTCATCTGTCATTGATGCACTGCAGTACAAATTAGAGGGCACCACAAGATTGA CAAGAAAAAGGGGATTGAAGTTAGCCACAGCTCTGTCTCTGAGCAACAAATTTGTGGAGGGTAGTCATAA CAGTACTGTGAGCTTAACCACGAAAAATATGGAAGTGTCAGTGGCAAAAACCACAAAAGCCGAAATTCCA ATTTTGAGAATGAATTTCAAGCAAGAACTTAATGGAAATACCAAGTCAAAACCTACTGTCTCTTCCTCCA TGGAATTTAAGTATGATTTCAATTCTTCAATGCTGTACTCTACCGCTAAAGGAGCAGTTGACCACAAGCT TAGCTTGGAAAGCCTCACCTCTTACTTTTCCATTGAGTCATCTACCAAAGGAGATGTCAAGGGTTCGGTT CTTTCTCGGGAATATTCAGGAACTATTGCTAGTGAGGCCAACACTTACTTGAATTCCAAGAGCACACGGT CTTCAGTGAAGCTGCAGGGCACTTCCAAAATTGATGATATCTGGAACCTTGAAGTAAAAGAAAATTTTGC TGGAGAAGCCACACTCCAACGCATATATTCCCTCTGGGAGCACAGTACGAAAAACCACTTACAGCTAGAG GGCCTCTTTTTCACCAACGGAGAACATACAAGCAAAGCCACCCTGGAACTCTCTCCATGGCAAATGTCAG CTCTTGTTCAGGTCCATGCAAGTCAGCCCAGTTCCTTCCATGATTTCCCTGACCTTGGCCAGGAAGTGGC CCTGAATGCTAACACTAAGAACCAGAAGATCAGATGGAAAAATGAAGTCCGGATTCATTCTGGGTCTTTC CAGAGCCAGGTCGAGCTTTCCAATGACCAAGAAAAGGCACACCTTGACATTGCAGGATCCTTAGAAGGAC ACCTAAGGTTCCTCAAAAATATCATCCTACCAGTCTATGACAAGAGCTTATGGGATTTCCTAAAGCTGGA TGTAACCACCAGCATTGGTAGGAGACAGCATCTTCGTGTTTCAACTGCCTTTGTGTACACCAAAAACCCC AATGGCTATTCATTCTCCATCCCTGTAAAAGTTTTGGCTGATAAATTCATTACTCCTGGGCTGAAACTAA ATGATCTAAATTCAGTTCTTGTCATGCCTACGTTCCATGTCCCATTTACAGATCTTCAGGTTCCATCGTG CAAACTTGACTTCAGAGAAATACAAATCTATAAGAAGCTGAGAACTTCATCATTTGCCCTCAACCTACCA ACACTCCCCGAGGTAAAATTCCCTGAAGTTGATGTGTTAACAAAATATTCTCAACCAGAAGACTCCTTGA TTCCCTTTTTTGAGATAACCGTGCCTGAATCTCAGTTAACTGTGTCCCAGTTCACGCTTCCAAAAAGTGT TTCAGATGGCATTGCTGCTTTGGATCTAAATGCAGTAGCCAACAAGATCGCAGACTTTGAGTTGCCCACC ATCATCGTGCCTGAGCAGACCATTGAGATTCCCTCCATTAAGTTCTCTGTACCTGCTGGAATTGTCATTC CTTCCTTTCAAGCACTGACTGCACGCTTTGAGGTAGACTCTCCCGTGTATAATGCCACTTGGAGTGCCAG TTTGAAAAACAAAGCAGATTATGTTGAAACAGTCCTGGATTCCACATGCAGCTCAACCGTACAGTTCCTA GAATATGAACTAAATGTTTTGGGAACACACAAAATCGAAGATGGTACGTTAGCCTCTAAGACTAAAGGAA CACTTGCACACCGTGACTTCAGTGCAGAATATGAAGAAGATGGCAAATTTGAAGGACTTCAGGAATGGGA AGGAAAAGCGCACCTCAATATCAAAAGCCCAGCGTTCACCGATCTCCATCTGCGCTACCAGAAAGACAAG AAAGGCATCTCCACCTCAGCAGCCTCCCCAGCCGTAGGCACCGTGGGCATGGATATGGATGAAGATGACG ACTTTTCTAAATGGAACTTCTACTACAGCCCTCAGTCCTCTCCAGATAAAAAACTCACCATATTCAAAAC TGAGTTGAGGGTCCGGGAATCTGATGAGGAAACTCAGATCAAAGTTAATTGGGAAGAAGAGGCAGCTTCT GGCTTGCTAACCTCTCTGAAAGACAACGTGCCCAAGGCCACAGGGGTCCTTTATGATTATGTCAACAAGT ACCACTGGGAACACACAGGGCTCACCCTGAGAGAAGTGTCTTCAAAGCTGAGAAGAAATCTGCAGAACAA TGCTGAGTGGGTTTATCAAGGGGCCATTAGGCAAATTGATGATATCGACGTGAGGTTCCAGAAAGCAGCC AGTGGCACCACTGGGACCTACCAAGAGTGGAAGGACAAGGCCCAGAATCTGTACCAGGAACTGTTGACTC AGGAAGGCCAAGCCAGTTTCCAGGGACTCAAGGATAACGTGTTTGATGGCTTGGTACGAGTTACTCAAAA ATTCCATATGAAAGTCAAGCATCTGATTGACTCACTCATTGATTTTCTGAACTTCCCCAGATTCCAGTTT CCGGGGAAACCTGGGATATACACTAGGGAGGAACTTTGCACTATGTTCATAAGGGAGGTAGGGACGGTAC TGTCCCAGGTATATTCGAAAGTCCATAATGGTTCAGAAATACTGTTTTCCTATTTCCAAGACCTAGTGAT TACACTTCCTTTCGAGTTAAGGAAACATAAACTAATAGATGTAATCTCGATGTATAGGGAACTGTTGAAA GATTTATCAAAAGAAGCCCAAGAGGTATTTAAAGCCATTCAGTCTCTCAAGACCACAGAGGTGCTACGTA ATCTTCAGGACCTTTTACAATTCATTTTCCAACTAATAGAAGATAACATTAAACAGCTGAAAGAGATGAA ATTTACTTATCTTATTAATTATATCCAAGATGAGATCAACACAATCTTCAATGATTATATCCCATATGTT TTTAAATTGTTGAAAGAAAACCTATGCCTTAATCTTCATAAGTTCAATGAATTTATTCAAAACGAGCTTC AGGAAGCTTCTCAAGAGTTACAGCAGATCCATCAATACATTATGGCCCTTCGTGAAGAATATTTTGATCC AAGTATAGTTGGCTGGACAGTGAAATATTATGAACTTGAAGAAAAGATAGTCAGTCTGATCAAGAACCTG TTAGTTGCTCTTAAGGACTTCCATTCTGAATATATTGTCAGTGCCTCTAACTTTACTTCCCAACTCTCAA GTCAAGTTGAGCAATTTCTGCACAGAAATATTCAGGAATATCTTAGCATCCTTACCGATCCAGATGGAAA AGGGAAAGAGAAGATTGCAGAGCTTTCTGCCACTGCTCAGGAAATAATTAAAAGCCAGGCCATTGCGACG AAGAAAATAATTTCTGATTACCACCAGCAGTTTAGATATAAACTGCAAGATTTTTCAGACCAACTCTCTG ATTACTATGAAAAATTTATTGCTGAATCCAAAAGATTGATTGACCTGTCCATTCAAAACTACCACACATT TCTGATATACATCACGGAGTTACTGAAAAAGCTGCAATCAACCACAGTCATGAACCCCTACATGAAGCTT GCTCCAGGAGAACTTACTATCATCCTCTAATTTTTTAAAAGAAATCTTCATTTATTCTTCTTTTCCAATT GAACTTTCACATAGCACAGAAAAAATTCAAACTGCCTATATTGATAAAACCATACAGTGAGCCAGCCTTG CAGTAGGCAGTAGACTATAAGCAGAAGCACATATGAACTGGACCTGCACCAAAGCTGGCACCAGGGCTCG GAAGGTCTCTGAACTCAGAAGGATGGCATTTTTTGCAAGTTAAAGAAAATCAGGATCTGAGTTATTTTGC TAAACTTGGGGGAGGAGGAACAAATAAATGGAGTCTTTATTGTGTATCATA (SEQ ID NO: 6681) >gi|4557442|ref|NM_000078.1| Homo sapiens cholesteryl ester transfer protein, plasma (CETP), mRNA GTGAATCTCTGGGGCCAGGAAGACCCTGCTGCCCGGAAGAGCCTCATGTTCCGTGGGGGCTGGGCGGACA TACATATACGGGCTCCAGGCTGAACGGCTCGGGCCACTTACACACCACTGCCTGATAACCATGCTGGCTG CCACAGTCCTGACCCTGGCCCTGCTGGGCAATGCCCATGCCTGCTCCAAAGGCACCTCGCACGAGGCAGG CATCGTGTGCCGCATCACCAAGCCTGCCCTCCTGGTGTTGAACCACGAGACTGCCAAGGTGATCCAGACC GCCTTCCAGCGAGCCAGCTACCCAGATATCACGGGCGAGAAGGCCATGATGCTCCTTGGCCAAGTCAAGT ATGGGTTGCACAACATCCAGATCAGCCACTTGTCCATCGCCAGCAGCCAGGTGGAGCTGGTGGAAGCCAA GTCCATTGATGTCTCCATTCAGAACGTGTCTGTGGTCTTCAAGGGGACCCTGAAGTATGGCTACACCACT GCCTGGTGGCTGGGTATTGATCAGTCCATTGACTTCGAGATCGACTCTGCCATTGACCTCCAGATCAACA CACAGCTGACCTGTGACTCTGGTAGAGTGCGGACCGATGCCCCTGACTGCTACCTGTCTTTCCATAAGCT GCTCCTGCATCTCCAAGGGGAGCGAGAGCCTGGGTGGATCAAGCAGCTGTTCACAAATTTCATCTCCTTC ACCCTGAAGCTGGTCCTGAAGGGACAGATCTGCAAAGAGATCAACGTCATCTCTAACATCATGGCCGATT TTGTCCAGACAAGGGCTGCCAGCATCCTTTCAGATGGAGACATTGGGGTGGACATTTCCCTGACAGGTGA TCCCGTCATCACAGCCTCCTACCTGGAGTCCCATCACAAGGGTCATTTCATCTACAAGAATGTCTCAGAG GACCTCCCCCTCCCCACCTTCTCGCCCACACTGCTGGGGGACTCCCGCATGCTGTACTTCTGGTTCTCTG AGCGAGTCTTCCACTCGCTGGCCAAGGTAGCTTTCCAGGATGGCCGCCTCATGCTCAGCCTGATGGGAGA CGAGTTCAAGGCAGTGCTGGAGACCTGGGGCTTCAACACCAACCAGGAAATCTTCCAAGAGGTTGTCGGC GGCTTCCCCAGCCAGGCCCAAGTCACCGTCCACTGCCTCAAGATGCCCAAGATCTCCTGCCAAAACAAGG GAGTCGTGGTCAATTCTTCAGTGATGGTGAAATTCCTCTTTCCACGCCCAGACCAGCAACATTCTGTAGC TTACACATTTGAAGAGGATATCGTGACTACCGTCCAGGCCTCCTATTCTAAGAAAAAGCTCTTCTTAAGC CTCTTGGATTTCCAGATTACACCAAAGACTGTTTCCAACTTGACTGAGAGCAGCTCCGAGTCCATCCAGA GCTTCCTGCAGTCAATGATCACCGCTGTGGGCATCCCTGAGGTCATGTCTCGGCTCGAGGTAGTGTTTAC AGCCCTCATGAACAGCAAAGGCGTGAGCCTCTTCGACATCATCAACCCTGAGATTATCACTCGAGATGGC TTCCTGCTGCTGCAGATGGACTTTGGCTTCCCTGAGCACCTGCTGGTGGATTTCCTCCAGAGCTTGAGCT AGAAGTCTCCAAGGAGGTCGGGATGGGGCTTGTAGCAGAAGGCAAGCACCAGGCTCACAGCTGGAACCCT GGTGTCTCCTCCAGCGTGGTGGAAGTTGGGTTAGGAGTACGGAGATGGAGATTGGCTCCCAACTCCTCCC TATCCTAAAGGCCCACTGGCATTAAAGTGCTGTATCCAAG (SEQ ID NO: 6682) >gi|414668|emb|X75500.1|HSMTP H.sapiens mRNA for microsomal triglyceride transfer protein TGCAGTTGAGGATTGCTGGTCAATATGATTCTTCTTGCTGTGCTTTTTCTCTGCTTCATTTCCTCATATT CAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGTACTC CACTGAAGTTCTTCTTGATCGGGGCAAAGGAAAACTGCAAGACAGCGTGGGCTACCGCATTTCCTCCAAC GTGGATGTGGCCTTACTATGGAGGAATCCTGATGGTGATGATGACCAGTTGATCCAAATAACGATGAAGG ATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGCATCTTCAAAGGAAAAAGCCCATCTAA AATAATGGGAAAGGAAAACTTGGAAGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAAGGTC AAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGAAAATATCAAGAGAGGTCTGGCTAGCCTAT TTCAGACACAGTTAAGCTCTGGAACCACCAATGAGGTAGATATCTCTGGAAATTGTAAAGTGACCTACCA GGCTCATCAAGACAAAGTGATCAAAATTAAGGCCTTGGATTCATGCAAAATAGCGAGGTCTGGATTTACG ACCCCAAATCAGGTCTTGGGTGTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCT TTGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCCTACAAACCATTAAGGGGAA AATAGTATCGAAGCAGAAATTAGAGCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAG GCTGCAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTGGGGCAGGTCTTCCAGA GCCACTGTAAAGGATGTCCTTCTCTCTCGGAGCTCTGGCGGTCCACCAGGAAATACCTGCAGCCTGACAA CCTTTCCAAGGCTGAGGCTGTCAGAAACTTCCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAA GAGATCCTTCAAATACTAAAGATGGAAAATAAGGAAGTATTACCTCAGCTGGTGGATGCTGTCACCTCTG CTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGGATTTCAAAAGTGACAGCAGCATTATCCT CCAGGAGAGGTTTCTCTATGCCTGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATT AGTAAGTTCAAAGGTTCTATTGGTAGCAGTGACATCAGAGAAACTGTTATGATCATCACTGGGACACTTG TCAGAAAGTTGTGTCAGAATGAAGGCTGCAAACTCAAAGCAGTAGTGGAAGCTAAGAAGTTAATCCTGGG AGGACTTGAAAAAGCAGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCCTGCTT CCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAAGGGCCCATCAGCCACCTGGCTACCA CTGCTCTCCAGAGATATGATCTCCCTTTCATAACTGATGAGGTGAAGAAGACCTTAAACAGAATATACCA CCAAAACCGTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTAAATAACAATCCA TCCTACATGGACGTCAAGAACATCCTGCTGTCTATTGGGGAGCTTCCCCAAGAAATGAATAAATACATGC TCGCCATTGTTCAAGACATCCTACGTTTTGAAATGCCTGCAAGCAAAATTGTCCGTCGAGTTCTGAAGGA AATGGTCGCTCACAATTATGACCGTTTCTCCAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGAA CGTAGTCCCCGTTCGGCATCTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGAA GTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAGCCAGGTGGTTATTGAAGCCCA AGGACTGGAAGCCTTAATCGCAGCCACCCCTGACGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTATG TCAGCCATCCTCTTTGATGTTCAGCTCAGACCTGTCACCTTTTTCAACGGATACAGTGATTTGATGTCCA AAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGGACTTATTCTGCTAATAGATCATTCTCA GGAACTTCAGTTACAATCTGGACTAAAAGCCAATATAGAGGTCCAGGGTGGTCTAGCTATTGATATTTCA GGTGCAATGGAGTTTAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAGGGTGACTGTGGTAA TAACCACTGACATCACAGTGGACTCCTCTTTTGTGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAGC AGGCTTGGAGTTTATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGATGGACAAG GATGAAGCTCCATTCAGGCAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTATGTCTCTC AGAAAAGAAAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCTCCATCAAGAGAACTCAGAGATGTGCAA AGTGGTGTTTGCCCCTCAGCCGGATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTAC TTGAATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTCTCTGAGAGCACAGCGTT TACATATTTACCTGTATTTAAGATTTTTGTAAAAAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGTA TATGCAGTATGCTACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTTAGTTTAC TTATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTATTCTCTAAGAGGAAACTAGTGTTTGTT AAAAACAAAAATAAAAACAAAACCACACAAGGAGAACCCAATTTTGTTTCAACAATTTTTGATCAATGTA TATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTTCCCTAATCAGGAAAAAA AAAAAAAAAAAGTAAGACACAAACAAACCATTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGGAG AAGGGACAAGGCTTTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTTATTGTTA GTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATC (SEQ ID NO: 6683) >gi|1217638|emb|X91148.1|HSMTTP H.sapiens mRNA for microsomal triglyceride transfer protein TGCAGTTGAGGATTGCTGGTCAATATGATTCTTCTTGCTGTGCTTTTTCTCTGCTTCATTTCCTCATATT CAGCTTCTGTTAAAGGTCACACAACTGGTCTCTCATTAAATAATGACCGGCTGTACAAGCTCACGTACTC CACTGAAGTTCTTCTTGATCGGGGCAAAGGAAAACTGCAAGACAGCGTGGGCTACCGCATTTCCTCCAAC GTGGATGTGGCCTTACTATGGAGGAATCCTGATGGTGATGATGACCAGTTGATCCAAATAACGATGAAGG ATGTAAATGTTGAAAATGTGAATCAGCAGAGAGGAGAGAAGAGCATCTTCAAAGGAAAAAGCCCATCTAA AATAATGGGAAAGGAAAACTTGGAAGCTCTGCAAAGACCTACGCTCCTTCATCTAATCCATGGAAAGGTC AAAGAGTTCTACTCATATCAAAATGAGGCAGTGGCCATAGAAAATATCAAGAGAGGTCTGGCTAGCCTAT TTCAGACACAGTTAAGCTCTGGAACCACCAATGAGGTAGATATCTCTGGAAATTGTAAAGTGACCTACCA GGCTCATCAAGACAAAGTGATCAAAATTAAGGCCTTGGATTCATGCAAAATAGCGAGGTCTGGATTTACG ACCCCAAATCAGGTCTTGGGTGTCAGTTCAAAAGCTACATCTGTCACCACCTATAAGATAGAAGACAGCT TTGTTATAGCTGTGCTTGCTGAAGAAACACACAATTTTGGACTGAATTTCCTACAAACCATTAAGGGGAA AATAGTATCGAAGCAGAAATTAGAGCTGAAGACAACCGAAGCAGGCCCAAGATTGATGTCTGGAAAGCAG GCTGCAGCCATAATCAAAGCAGTTGATTCAAAGTACACGGCCATTCCCATTGTGGGGCAGGTCTTCCAGA GCCACTGTAAAGGATGTCCTTCTCTCTCGGAGCTCTGGCGGTCCACCAGGAAATACCTGCAGCCTGACAA CCTTTCCAAGGCTGAGGCTGTCAGAAACTTCCTGGCCTTCATTCAGCACCTCAGGACTGCGAAGAAAGAA GAGATCCTTCAAATACTAAAGATGGAAAATAAGGAAGTATTACCTCAGCTGGTGGATGCTGTCACCTCTG CTCAGACCTCAGACTCATTAGAAGCCATTTTGGACTTTTTGGATTTCAAAAGTGACAGCAGCATTATCCT CCAGGAGAGGTTTCTCTATGCCTGTGGATTTGCTTCTCATCCCAATGAAGAACTCCTGAGAGCCCTCATT AGTAAGTTCAAAGGTTCTATTGGTAGCAGTGACATCAGAGAAACTGTTATGATCATCACTGGGACACTTG TCAGAAAGTTGTGTCAGAATGAAGGCTGCAAACTCAAAGCAGTAGTGGAAGCTAAGAAGTTAATCCTGGG AGGACTTGAAAAAGCAGAGAAAAAAGAGGACACCAGGATGTATCTGCTGGCTTTGAAGAATGCCCTGCTT CCAGAAGGCATCCCAAGTCTTCTGAAGTATGCAGAAGCAGGAGAAGGGCCCATCAGCCACCTGGCTACCA CTGCTCTCCAGAGATATGATGCTCCCTTTCATAaCTGATGAGGTGAAGAAGACCTTAAACAGAATATACC ACCAAAACCGTAAAGTTCATGAAAAGACTGTGCGCACTGCTGCAGCTGCTATCATTTTAAATAACAATCC ATCCTACATGGACGTCAAGAACATCCTGCTGTCTATTGGGGAGCTTCCCCAAGAAATGAATAAATACATG CTCGCCATTGTTCAAGACATCCTACGTTTTGAAATGCCTGCAAGCAAAATTGTCCGTCGAGTTCTGAAGG AAATGGTCGCTCACAATTATGACCGTTTCTCCAGGAGTGGATCTTCTTCTGCCTACACTGGCTACATAGA ACGTAGTCCCCGTTCGGCATCTACTTACAGCCTAGACATTCTCTACTCGGGTTCTGGCATTCTAAGGAGA AGTAACCTGAACATCTTTCAGTACATTGGGAAGGCTGGTCTTCACGGTAGCCAGGTGGTTATTGAAGCCC AAGGACTGGAAGCCTTAATCGCAGCCACCCCTGACGAGGGGGAGGAGAACCTTGACTCCTATGCTGGTAT GTCAGCCATCCTCTTTGATGTTCAGCTCAGACCTGTCACCTTTTTCAACGGATACAGTGATTTGATGTCC AAAATGCTGTCAGCATCTGGCGACCCTATCAGTGTGGTGAAAGGACTTATTCTGCTAATAGATCATTCTC AGGAACTTCAGTTACAATCTGGACTAAAAGCCAATATAGAGGTCCAGGGTGGTCTAGCTATTGATATTTC AGGTGCAATGGAGTTTAGCTTGTGGTATCGTGAGTCTAAAACCCGAGTGAAAAATAGGGTGACTGTGGTA ATAACCACTGACATCACAGTGGACTCCTCTTTTGTGAAAGCTGGCCTGGAAACCAGTACAGAAACAGAAG CAGGCTTGGAGTTTATCTCCACAGTGCAGTTTTCTCAGTACCCATTCTTAGTTTGCATGCAGATGGACAA GGATGAAGCTCCATTCAGGCAATTTGAGAAAAAGTACGAAAGGCTGTCCACAGGCAGAGGTTATGTCTCT CAGAAAAGAAAAGAAAGCGTATTAGCAGGATGTGAATTCCCGCTCCATCAAGAGAACTCAGAGATGTGCA AAGTGGTGTTTGCCCCTCAGCCGGATAGTACTTCCAGCGGATGGTTTTGAAACTGACCTGTGATATTTTA CTTGAATTTGTCTCCCCGAAAGGGACACAATGTGGCATGACTAAGTACTTGCTCTCTGAGAGCACAGCGT TTACATATTTACCTGTATTTAAGATTTTTGTAAAAAGCTACAAAAAACTGCAGTTTGATCAAATTTGGGT ATATGCAGTATGCTACCCACAGCGTCATTTTGAATCATCATGTGACGCTTTCAACAACGTTCTTAGTTTA CTTATACCTCTCTCAAATCTCATTTGGTACAGTCAGAATAGTTATTCTCTAAGAGGAAACTAGTGTTTGT TAAAAACAAAAATAAAAACAAAACCACACAAGGAGAACCCAATTTTGTTTCAACAATTTTTGATCAATGT ATATGAAGCTCTTGATAGGACTTCCTTAAGCATGACGGGAAAACCAAACACGTTCCCTAATCAGGAAAAA AAAAAAAAAAGAAAAAGTAAGACACAAACAAACCATTTTTTTCTCTTTTTTTGGAGTTGGGGGCCCAGGG AGAAGGGACAAGGCTTTTAAAAGACTTGTTAGCCAACTTCAAGAATTAATATTTATGTCTCTGTTATTGT TAGTTTTAAGCCTTAAGGTAGAAGGCACATAGAAATAACATCTCATCTTTCTGCTGACCATTTTAGTGAG GTTGTTCCAAAGAGCATTCAGGTCTCTACCTCCAGCCCTGCAAAAATATTGGACCTAGCACAGAGGAATC AGGAAAATTAATTTCAGAAACTCCATTTGATTTTTCTTTTGCTGTGTCTTTTTTGAGACTGTAATATGGT ACACTGTCCTCTAAGGACATCCTCATTTTATCTCACCTTTTTGGGGGTGAGAGCTCTAGTTCATTTAACT GTACTCTGCACAATAGCTAGGATGACTAAGAGAACATTGCTTCAAGAAACTGGTGGATTTGGATTTCCAA AATATGAAATAAGGAGAAAAATGTTTTTATTTGTATGAATTAAAAGATCCATGTTGAACATTTGCAAATA TTTATTAATAAACAGATGTGGTGATAAACCCAAAACAAATGACAGGTGCTTATTTTCCACTAAACACAGA CACATGAAATGAAAGTTTAGCTAGCCCACTATTTGTTGTAAATTGAAAACGAAGTGTGATAAAATAAATA TGTAGAAATC (SEQ ID NO: 6684) >gi|21361125|ref|NM_001467.2| Homo sapiens glucose-6-phosphatase, transport (glucose-6-phosphate) protein 1 (G6PT1), mRNA GGCACGAGGGGCCACCGAGGCGCTGTCCCTGACCACCAGCACGAGACCCCTTTCTATCGCGCCAGTCCTG TGGTCTCCGCACCTCTCCAGCTCCTGCACCCCCGGCCCCCGTGGTTCCCAGCCGCACAGTAGCGTGTCCT GGGTAGCGTGAGGACCCACGGGGCTGAGCAGGTGCCACGAGCCCGCCGCCTCTTCGCCGCCCGCCGCCTC TCCTCCTCTCCCGCCCGCCGCCTGGCCCTCCCCTACCAGGCTGAGCCTCTGGCTGCCAGAAGCGCGGGGC CTCCGGGAGAATACGTGCGGTCGCCCGCTCCGCGTGCGCCTACGCCTTCTGCTCCAGTTGCTTTCCCAAT TGAGCGGAAAAGCCGGGGCATGTTGCCGGGGCCCTGGGCGGGACGGTTGTGCCCTGCAGCCCGAAGCCCG CCGGGGCACCTTCCCGCCCACGAGCTGCCCAGTCCCTCTGCTTGCGGCCCCTGCCAACGTCCCACAGGAC ACTGGGTCCCCTTGGAGCCTCCCCAGGCTTAATGATTGTCCAGAAGGCGGCTATAAAGGGAGCCTGGGAG GCTGGGTGGAGGAGGGAGCAGAAAAAACCCAACTCAGCAGATCTGGGAACTGTGAGAGCGGCAAGCAGGA ACTGTGGTCAGAGGCTGTGCGTCTTGGCTGGTAGGGCCTGCTCTTTTCTACCATGGCAGCCCAGGGCTAT GGCTATTATCGCACTGTGATCTTCTCAGCCATGTTTGGGGGCTACAGCCTGTATTACTTCAATCGCAAGA CCTTCTCCTTTGTCATGCCATCATTGGTGGAAGAGATCCCTTTGGACAAGGATGATTTGGGGTTCATCAC CAGCAGCCAGTCGGCAGCTTATGCTATCAGCAAGTTTGTCAGTGGGGTGCTGTCTGACCAGATGAGTGCT CGCTGGCTCTTCTCTTCTGGGCTGCTCCTGGTTGGCCTGGTCAACATATTCTTTGCCTGGAGCTCCACAG TACCTGTCTTTGCTGCCCTCTGGTTCCTTAATGGCCTGGCCCAGGGGCTGGGCTGGCCCCCATGTGGGAA GGTCCTGCGGAAGTGGTTTGAGCCATCTCAGTTTGGCACTTGGTGGGCCATCCTGTCAACCAGCATGAAC CTGGCTGGAGGGCTGGGCCCTATCCTGGCAACCATCCTTGCCCAGAGCTACAGCTGGCGCAGCACGCTGG CCCTATCTGGGGCACTGTGTGTGGTTGTCTCCTTCCTCTGTCTCCTGCTCATCCACAATGAACCTGCTGA TGTTGGACTCCGCAACCTGGACCCCATGCCCTCTGAGGGCAAGAAGGGCTCCTTGAAGGAGGAGAGCACC CTGCAGGAGCTGCTGCTGTCCCCTTACCTGTGGGTGCTCTCCACTGGTTACCTTGTGGTGTTTGGAGTAA AGACCTGCTGTACTGACTGGGGCCAGTTCTTCCTTATCCAGGAGAAAGGACAGTCAGCCCTTGTAGGTAG CTCCTACATGAGTGCCCTGGAAGTTGGGGGCCTTGTAGGCAGCATCGCAGCTGGCTACCTGTCAGACCGG GCCATGGCAAAGGCGGGACTGTCCAACTACGGGAACCCTCGCCATGGCCTGTTGCTGTTCATGATGGCTG GCATGACAGTGTCCATGTACCTCTTCCGGGTAACAGTGACCAGTGACTCCCCCAAGCTCTGGATCCTGGT ATTGGGAGCTGTATTTGGTTTCTCCTCGTATGGCCCCATTGCCCTGTTTGGAGTCATAGCCAACGAGAGT GCCCCTCCCAACTTGTGTGGCACCTCCCACGCCATTGTGGGACTCATGGCCAATGTGGGCGGCTTTCTGG CTGGGCTGCCCTTCAGCACCATTGCCAAGCACTACAGTTGGAGCACAGCCTTCTGGGTGGCTGAAGTGAT TTGTGCGGCCAGCACGGCTGCCTTCTTCCTCCTACGAAACATCCGCACCAAGATGGGCCGAGTGTCCAAG AAGGCTGAGTGAAGAGAGTCCAGGTTCCGGAGCACCATCCCACGGTGGCCTTCCCCCTGCACGCTCTGCG GGGAGAAAAGGAGGGGCCTGCCTGGCTAGCCCTGAACCTTTCACTTTCCATTTCTGCGCCTTTTCTGTCA CCCGGGTGGCGCTGGAAGTTATCAGTGGCTAGTGAGGTCCCAGCTCCCTGATCCTATGCTCTATTTAAAA GATAACCTTTGGCCTTAGACTCCGTTAGCTCCTATTTCCTGCCTTCAGACAAACAGGAAACTTCTGCAGT CAGGAAGGCTCCTGTACCCTTCTTCTTTTCCTAGGCCCTGTCCTGCCCGCATCCTACCCCATCCCCACCT GAAGTGAGGCTATCCCTGCAGCTGCAGGGCACTAATGACCCTTGACTTCTGCTGGGTCCTAAGTCCTCTC AGCAGTGGGTGACTGCTGTTGCCAATACCTCAGACTCCAGGGAAAGAGAGGAGGCCATCATTCTCACTGT ACCACTAGGCGCAGTTGGATATAGGTGGGAAGAAAAGGTGACTTGTTATAGAAGATTAAAACTAGATTTG ATACTG (SEQ ID NO: 6685) gi|4503130|ref|NM_001904.1| Homo sapiens catenin (cadherin-associated protein), beta 1, 88kDa (CTNNB1), mRNA AAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCCGGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGAGACGG AGGAAGGTCTGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGGACGGTCGGACTCCCG CGGCGGGAGGAGCCTGTTCCCCTGAGGGTATTTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTG GACAATGGCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGACAGAAAAGCGGCTGTT AGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCCACTACCACAGCTCCTTCTC TGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAAGTCCTGTATGAGTGGGAACAGGG ATTTTCTCAGTCCTTCACTCAAGAACAAGTAGCTGATATTGATGGACAGTATGCAATGACTCGAGCTCAG AGGGTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGGGCATGCAGATCCCATCTACACAGTTTGATG CTGCTCATCCCACTAATGTCCAGCGTTTGGCTGAACCATCACAGATGCTGAAACATGCAGTTGTAAACTT GATTAACTATCAAGATGATGCAGAACTTGCCACACGTGCAATCCCTGAACTGACAAAACTGCTAAATGAC GAGGACCAGGTGGTGGTTAATAAGGCTGCAGTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCCAGAC ACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCTATTGTACGTACCATGCAGAATACAAATGATGTAGA AACAGCTCGTTGTACCGCTGGGACCTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCCATCTTT AAGTCTGGAGGCATTCCTGCCCTGGTGAAAATGCTTGGTTCACCAGTGGATTCTGTGTTGTTTTATGCCA TTACAACTCTCCACAACCTTTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTGGTGGGCT GCAGAAAATGGTTGCCTTGCTCAACAAAACAAATGTTAAATTCTTGGCTATTACGACAGACTGCCTTCAA ATTTTAGCTTATGGCAACCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAAGCTTTAGTAA ATATAATGAGGACCTATACTTACGAAAAACTACTGTGGACCACAAGCAGAGTGCTGAAGGTGCTATCTGT CTGCTCTAGTAATAAGCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTTCACCTGACAGAT CCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGACTCTCAGGAATCTTTCAGATGCTGCAACTAAACAGG AAGGGATGGAAGGTCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATAAATGTGGTCACCTG TGCAGCTGGAATTCTTTCTAACCTCACTTGCAATAATTATAAGAACAAGATGATGGTCTGCCAAGTGGGT GGTATAGAGGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCT GTGCTCTTCGTCATCTGACCAGCCGACACCAAGAAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTA TGGACTACCAGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTGATAAAGGCTACTGTTGGA TTGATTCGAAATCTTGCCCTTTGTCCCGCAAATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGAC TAGTTCAGTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCCATGGGTGGGACACAGCAGCA ATTTGTGGAGGGGGTCCGCATGGAAGAAATAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGG GATGTTCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTGTTTGTGCAGCTGCTTTATTCTC CCATTGAAAACATCCAAAGAGTAGCTGCAGGGGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGA AGCTATTGAAGCTGAGGGAGCCACAGCTCCTCTGACAGAGTTACTTCACTCTAGGAATGAAGGTGTGGCG ACATATGCAGCTGCTGTTTTGTTCCGAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTCAG TTGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGGAATGAGACTGCTGATCTTGGACTTGA TATTGGTGCCCAGGGAGAACCCCTTGGATATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTCTGGT GGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGAACATGAGATGGGTGGCCACCACCCTGGTG CTGACTATCCAGTTGATGGGCTGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCCTCCAGG TGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCCTTTAGCTGTATTGTCTGAACTTG CATTGTGATTGGCCTGTAGAGTTGCTGAGAGGGCTCGAGGGGTGGGCTGGTATCTCAGAAAGTGCCTGAC ACACTAACCAAGCTGAGTTTCCTATGGGAACAATTGAAGTAAACTTTTTGTTCTGGTCCTTTTTGGTCGA GGAGTAACAATACAAATGGATTTTGGGAGTGACTCAAGAAGTGAAGAATGCACAAGAATGGATCACAAGA TGGAATTTAGCAAACCCTAGCCTTGCTTGTTAAAATTTTTTTTTTTTTTTTTTTAAGAATATCTGTAATG GTACTGACTTTGCTTGCTTTGAAGTAGCTCTTTTTTTTTTTTTTTTTTTTTTTTTTTGCAGTAACTGTTT TTTAAGTCTCTCGTAGTGTTAAGTTATAGTGAATACTGCTACAGCAATTTCTAATTTTTAAGAATTGAGT AATGGTGTAGAACACTAATTAATTCATAATCACTCTAATTAATTGTAATCTGAATAAAGTGTAACAATTG TGTAGCCTTTTTGTATAAAATAGACAAATAGAAAATGGTCCAATTAGTTTCCTTTTTAATATGCTTAAAA TAAGCAGGTGGATCTATTTCATGTTTTTGATCAAAAACTATTTGGGATATGTATGGGTAGGGTAAATCAG TAAGAGGTGTTATTTGGAACCTTGTTTTGGACAGTTTACCAGTTGCCTTTTATCCCAAAGTTGTTGTAAC CTGCTGTGATACGATGCTTCAAGAGAAAATGCGGTTATAAAAAATGGTTCAGAATTAAACTTTTAATTCA TT (SEQ ID NO: 6686) gi|18104977|ref|NM_002827.2| Homo sapiens protein tyrosine phosphatase, non-receptor type 1 (PTPN1), mRNA GTGATGCGTAGTTCCGGCTGCCGGTTGACATGAAGAAGCAGCAGCGGCTAGGGCGGCGGTAGCTGCAGGG GTCGGGGATTGCAGCGGGCCTCGGGGCTAAGAGCGCGACGCGGCCTAGAGCGGCAGACGGCGCAGTGGGC CGAGAAGGAGGCGCAGCAGCCGCCCTGGCCCGTCATGGAGATGGAAAAGGAGTTCGAGCAGATCGACAAG TCCGGGAGCTGGGCGGCCATTTACCAGGATATCCGACATGAAGCCAGTGACTTCCCATGTAGAGTGGCCA AGCTTCCTAAGAACAAAAACCGAAATAGGTACAGAGACGTCAGTCCCTTTGACCATAGTCGGATTAAACT ACATCAAGAAGATAATGACTATATCAACGCTAGTTTGATAAAAATGGAAGAAGCCCAAAGGAGTTACATT CTTACCCAGGGCCCTTTGCCTAACACATGCGGTCACTTTTGGGAGATGGTGTGGGAGCAGAAAAGCAGGG GTGTCGTCATGCTCAACAGAGTGATGGAGAAAGGTTCGTTAAAATGCGCACAATACTGGCCACAAAAAGA AGAAAAAGAGATGATCTTTGAAGACACAAATTTGAAATTAACATTGATCTCTGAAGATATCAAGTCATAT TATACAGTGCGACAGCTAGAATTGGAAAACCTTACAACCCAAGAAACTCGAGAGATCTTACATTTCCACT ATACCACATGGCCTGACTTTGGAGTCCCTGAATCACCAGCCTCATTCTTGAACTTTCTTTTCAAAGTCCG AGAGTCAGGGTCACTCAGCCCGGAGCACGGGCCCGTTGTGGTGCACTGCAGTGCAGGCATCGGCAGGTCT GGAACCTTCTGTCTGGCTGATACCTGCCTCTTGCTGATGGACAAGAGGAAAGACCCTTCTTCCGTTGATA TCAAGAAAGTGCTGTTAGAAATGAGGAAGTTTCGGATGGGGCTGATCCAGACAGCCGACCAGCTGCGCTT CTCCTACCTGGCTGTGATCGAAGGTGCCAAATTCATCATGGGGGACTCTTCCGTGCAGGATCAGTGGAAG GAGCTTTCCCACGAGGACCTGGAGCCCCCACCCGAGCATATCCCCCCACCTCCCCGGCCACCCAAACGAA TCCTGGAGCCACACAATGGGAAATGCAGGGAGTTCTTCCCAAATCACCAGTGGGTGAAGGAAGAGACCCA GGAGGATAAAGACTGCCCCATCAAGGAAGAAAAAGGAAGCCCCTTAAATGCCGCACCCTACGGCATCGAA AGCATGAGTCAAGACACTGAAGTTAGAAGTCGGGTCGTGGGGGGAAGTCTTCGAGGTGCCCAGGCTGCCT CCCCAGCCAAAGGGGAGCCGTCACTGCCCGAGAAGGACGAGGACCATGCACTGAGTTACTGGAAGCCCTT CCTGGTCAACATGTGCGTGGCTACGGTCCTCACGGCCGGCGCTTACCTCTGCTACAGGTTCCTGTTCAAC AGCAACACATAGCCTGACCCTCCTCCACTCCACCTCCACCCACTGTCCGCCTCTGCCCGCAGAGCCCACG CCCGACTAGCAGGCATGCCGCGGTAGGTAAGGGCCGCCGGACCGCGTAGAGAGCCGGGCCCCGGACGGAC GTTGGTTCTGCACTAAAACCCATCTTCCCCGGATGTGTGTCTCACCCCTCATCCTTTTACTTTTTGCCCC TTCCACTTTGAGTACCAAATCCACAAGCCATTTTTTGAGGAGAGTGAAAGAGAGTACCATGCTGGCGGCG CAGAGGGAAGGGGCCTACACCCGTCTTGGGGCTCGCCCCACCCAGGGCTCCCTCCTGGAGCATCCCAGGC GGGCGGCACGCCAACAGCCCCCCCCTTGAATCTGCAGGGAGCAACTCTCCACTCCATATTTATTTAAACA ATTTTTTCCCCAAAGGCATCCATAGTGCACTAGCATTTTCTTGAACCAATAATGTATTAAAATTTTTTGA TGTCAGCCTTGCATCAAGGGCTTTATCAAAAAGTACAATAATAAATCCTCAGGTAGTACTGGGAATGGAA GGCTTTGCCATGGGCCTGCTGCGTCAGACCAGTACTGGGAAGGAGGACGGTTGTAAGCAGTTGTTATTTA GTGATATTGTGGGTAACGTGAGAAGATAGAACAATGCTATAATATATAATGAACACGTGGGTATTTAATA AGAAACATGATGTGAGATTACTTTGTCCCGCTTATTCTCCTCCCTGTTATCTGCTAGATCTAGTTCTCAA TCACTGCTCCCCCGTGTGTATTAGAATGCATGTAAGGTCTTCTTGTGTCCTGATGAAAAATATGTGCTTG AAATGAGAAACTTTGATCTCTGCTTACTAATGTGCCCCATGTCCAAGTCCAACCTGCCTGTGCATGACCT GATCATTACATGGCTGTGGTTCCTAAGCCTGTTGCTGAAGTCATTGTCGCTCAGCAATAGGGTGCAGTTT TCCAGGAATAGGCATTTGCCTAATTCCTGGCATGACACTCTAGTGACTTCCTGGTGAGGCCCAGCCTGTC CTGGTACAGCAGGGTCTTGCTGTAACTCAGACATTCCAAGGGTATGGGAAGCCATATTCACACCTCACGC TCTGGACATGATTTAGGGAAGCAGGGACACCCCCCGCCCCCCACCTTTGGGATCAGCCTCCGCCATTCCA AGTCAACACTCTTCTTGAGCAGACCGTGATTTGGAAGAGAGGCACCTGCTGGAAACCACACTTCTTGAAA CAGCCTGGGTGACGGTCCTTTAGGCAGCCTGCCGCCGTCTCTGTCCCGGTTCACCTTGCCGAGAGAGGCG CGTCTGCCCCACCCTCAAACCCTGTGGGGCCTGATGGTGCTCACGACTCTTCCTGCAAAGGGAACTGAAG ACCTCCACATTAAGTGGCTTTTTAACATGAAAAACACGGCAGCTGTAGCTCCCGAGCTACTCTCTTGCCA GCATTTTCACATTTTGCCTTTCTCGTGGTAGAAGCCAGTACAGAGAAATTCTGTGGTGGGAACATTCGAG GTGTCACCCTGCAGAGCTATGGTGAGGTGTGGATAAGGCTTAGGTGCCAGGCTGTAAGCATTCTGAGCTG GGCTTGTTGTTTTTAAGTCCTGTATATGTATGTAGTAGTTTGGGTGTGTATATATAGTAGCATTTCAAAA TGGACGTACTGGTTTAACCTCCTATCCTTGGAGAGCAGCTGGCTCTCCACCTTGTTACACATTATGTTAG AGAGGTAGCGAGCTGCTCTGCTATATGCCTTAAGCCAATATTTACTCATCAGGTCATTATTTTTTACAAT GGCCATGGAATAAACCATTTTTACAAAA (SEQ ID NO: 6687) gi|12831192|gb|AF333324.1| Hepatitis C virus type 1b polyprotein mRNA, complete cds GCCAGCCCCCGATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCA GAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCA TAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCG CTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCC TTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCATCATGAGCACA AATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGACGTTAAGTTCCCGGGCG GTGGTCAGATCGTTGGTGGAGTTTACCTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAG GAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGGCCCGAGGGT AGGACCTGGGCTCAGCCCGGGTACCCTTGGCCCCTCTATGGCAACGAGGGTATGGGGTGGGCAGGATGGC TCCTGTCACCCCGTGGCTCTCGGCCTAGTTGGGGCCCCACAGACCCCCGGCGTAGGTCGCGTAATTTGGG TAAGGTCATCGATACCCTTACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTTGTCGGCGCCCCC CTAGGAGGCGCTGCCAGGGCCCTGGCGCATGGCGTCCGGGTTCTGGAGGACGGCGTGAACTATGCAACAG GGAATCTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTAGCTTTGCTGTCTTGTTTGACCATCCCAGCTTC CGCTTACGAGGTGCGCAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGTATTGTG TATGAGGCAGCGGACATGATCATGCACACCCCCGGGTGCGTGCCCTGCGTCCGGGAGAGTAATTTCTCCC GTTGCTGGGTAGCGCTCACTCCCACGCTCGCGGCCAGGAACAGCAGCATCCCCACCACGACAATACGACG CCACGTCGATTTGCTCGTTGGGGCGGCTGCTCTCTGTTCCGCTATGTACGTTGGGGATCTCTGCGGATCC GTTTTTCTCGTCTCCCAGCTGTTCACCTTCTCACCTCGCCGGTATGAGACGGTACAAGATTGCAATTGCT CAATCTATCCCGGCCACGTATCAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCACCTACAAC GGCCCTAGTGGTATCGCAGCTACTCCGGATCCCACAAGCCGTCGTGGACATGGTGGCGGGGGCCCACTGG GGTGTCCTAGCGGGCCTTGCCTACTATTCCATGGTGGGGAACTGGGCTAAGGTCTTGATTGTGATGCTAC TCTTTGCTGGCGTTGACGGGCACACCCACGTGACAGGGGGAAGGGTAGCCTCCAGCACCCAGAGCCTCGT GTCCTGGCTCTCACAAGGGCCATCTCAGAAAATCCAACTCGTGAACACCAACGGCAGCTGGCACATCAAC AGGACCGCTCTGAATTGCAATGACTCCCTCCAAACTGGGTTCATTGCTGCGCTGTTCTACGCACACAGGT TCAACGCGTCCGGATGTCCAGAGCGCATGGCCAGCTGCCGCCCCATCGACAAGTTCGCTCAGGGGTGGGG TCCCATCACTCACGTTGTGCCTAACATCTCGGACCAGAGGCCTTATTGCTGGCACTATGCACCCCAACCG TGCGGTATTGTACCCGCGTCGCAGGTGTGTGGCCCAGTGTATTGCTTCACCCCGAGTCCTGTTGTGGTGG GGACGACCGACCGTTCCGGAGTCCCCACGTATAGCTGGGGGGAGAATGAGACAGACGTGCTGCTACTCAA CAACACGCGGCCGCCGCAAGGCAACTGGTTCGGCTGTACATGGATGAATAGCACCGGGTTCACCAAGACG TGCGGGGGCCCCCCGTGTAACATCGGGGGGGTTGGCAACAACACCTTGATTTGCCCCACGGATTGCTTCC GAAAGCACCCCGAGGCCACTTACACCAAATGCGGCTCGGGTCCTTGGTTGACACCTAGGTGTCTAGTTGA CTACCCATACAGACTTTGGCACTACCCCTGCACTATCAATTTTACCATCTTCAAGGTCAGGATGTACGTG GGGGGCGTGGAGCACAGGCTCAACGCCGCGTGCAATTGGACCCGAGGAGAGCGCTGTGACCTGGAGGACA GGGATAGATCAGAGCTTAGCCCGCTGCTATTGTCTACAACGGAGTGGCAGGTACTGCCCTGTTCCTTTAC CACCCTACCGGCTCTGTCCACTGGATTGATCCACCTCCATCAGAATATCGTGGACGTGCAATACCTGTAC GGTGTAGGGTCAGTGGTTGTCTCCGTCGTAATCAAATGGGAGTATGTTCTGCTGCTCTTCCTTCTCCTGG CGGACGCGCGCGTCTGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCTGAGGCCACCTTAGAGAA CCTGGTGGTCCTCAATGCGGCGTCTGTGGCCGGAGCGCATGGCCTTCTCTCCTTCCTCGTGTTCTTCTGC GCCGCCTGGTACATCAAAGGCAGGCTGGTCCCTGGGGCGGCATATGCTCTCTATGGCGTATGGCCGTTGC TCCTGCTCTTGCTGGCTTTACCACCACGAGCTTATGCCATGGACCGAGAGATGGCTGCATCGTGCGGAGG CGCGGTTTTTGTAGGTCTGGTACTCTTGACCTTGTCACCATACTATAAGGTGTTCCTCGCTAGGCTCATA TGGTGGTTACAATATTTTATCACCAGGGCCGAGGCGCACTTGCAAGTGTGGGTCCCCCCTCTTAATGTTC GGGGAGGCCGCGATGCCATCATCCTCCTTACATGCGCGGTCCATCCAGAGCTAATCTTTGACATCACCAA ACTCCTGCTCGCCATACTCGGTCCGCTCATGGTGCTCCAAGCTGGCATAACCAGAGTGCCGTACTTCGTG CGCGCTCAAGGGCTCATTCATGCATGCATGTTAGTGCGGAAGGTCGCTGGGGGTCATTATGTCCAAATGG CCTTCATGAAGCTGGGCGCGCTGACAGGCACGTACATTTACAACCATCTTACCCCGCTACGGGATTGGGC CCACGCGGGCCTACGAGACCTTGCGGTGGCAGTGGAGCCCGTCGTCTTCTCCGACATGGAGACCAAGATC ATCACCTGGGGAGCAGACACCGCGGCGTGTGGGGACATCATCTTGGGTCTGCCCGTCTCCGCCCGAAGGG GAAAGGAGATACTCCTGGGCCCGGCCGATAGTCTTGAAGGGCGGGGGTGGCGACTCCTCGCGCCCATCAC GGCCTACTCCCAACAGACGCGGGGCCTACTTGGTTGCATCATCACTAGCCTTACAGGCCGGGACAAGAAC CAGGTCGAGGGAGAGGTTCAGGTGGTTTCCACCGCAACACAATCCTTCCTGGCGACCTGCGTCAACGGCG TGTGTTGGACCGTTTACCATGGTGCTGGCTCAAAGACCTTAGCCGGCCCAAAGGGGCCAATCACCCAGAT GTACACTAATGTGGACCAGGACCTCGTCGGCTGGCAGGCGCCCCCCGGGGCGCGTTCCTTGACACCATGC ACCTGTGGCAGCTCAGACCTTTACTTGGTCACGAGACATGCTGACGTCATTCCGGTGCGCCGGCGGGGCG ACAGTAGGGGGAGCCTGCTCTCCCCCAGGCCTGTCTCCTACTTGAAGGGCTCTTCGGGTGGTCCACTGCT CTGCCCTTCGGGGCACGCTGTGGGCATCTTCCGGGCTGCCGTATGCACCCGGGGGGTTGCGAAGGCGGTG GACTTTGTGCCCGTAGAGTCCATGGAAACTACTATGCGGTCTCCGGTCTTCACGGACAACTCATCCCCCC CGGCCGTACCGCAGTCATTTCAAGTGGCCCACCTACACGCTCCCACTGGCAGCGGCAAGAGTACTAAAGT GCCGGCTGCATATGCAGCCCAAGGGTACAAGGTGCTCGTCCTCAATCCGTCCGTTGCCGCTACCTTAGGG TTTGGGGCGTATATGTCTAAGGCACACGGTATTGACCCCAACATCAGAACTGGGGTAAGGACCATTACCA CAGGCGCCCCCGTCACATACTCTACCTATGGCAAGTTTCTTGCCGATGGTGGTTGCTCTGGGGGCGCTTA TGACATCATAATATGTGATGAGTGCCATTCAACTGACTCGACTACAATCTTGGGCATCGGCACAGTCCTG GACCAAGCGGAGACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACCGCTACGCCTCCGGGATCGGTCACCG TGCCACACCCAAACATCGAGGAGGTGGCCCTGTCTAATACTGGAGAGATCCCCTTCTATGGCAAAGCCAT CCCCATTGAAGCCATCAGGGGGGGAAGGCATCTCATTTTCTGTCATTCCAAGAAGAAGTGCGACGAGCTC GCCGCAAAGCTGTCAGGCCTCGGAATCAACGCTGTGGCGTATTACCGGGGGCTCGATGTGTCCGTCATAC CAACTATCGGAGACGTCGTTGTCGTGGCAACAGACGCTCTGATGACGGGCTATACGGGCGACTTTGACTC AGTGATCGACTGTAACACATGTGTCACCCAGACAGTCGACTTCAGCTTGGATCCCACCTTCACCATTGAG ACGACGACCGTGCCTCAAGACGCAGTGTCGCGCTCGCAGCGGCGGGGTAGGACTGGCAGGGGTAGGAGAG GCATCTACAGGTTTGTGACTCCGGGAGAACGGCCCTCGGGCATGTTCGATTCCTCGGTCCTGTGTGAGTG CTATGACGCGGGCTGTGCTTGGTACGAGCTCACCCCCGCCGAGACCTCGGTTAGGTTGCGGGCCTACCTG AACACACCAGGGTTGCCCGTTTGCCAGGACCACCTGGAGTTCTGGGAGAGTGTCTTCACAGGCCTCACCC ACATAGATGCACACTTCTTGTCCCAGACCAAGCAGGCAGGAGACAACTTCCCCTACCTGGTAGCATACCA AGCCACGGTGTGCGCCAGGGCTCAGGCCCCACCTCCATCATGGGATCAAATGTGGAAGTGTCTCATACGG CTGAAACCTACGCTGCACGGGCCAACACCCTTGCTGTACAGGCTGGGAGCCGTCCAAAATGAGGTCACCC TCACCCACCCCATAACCAAATACATCATGGCATGCATGTCGGCTGACCTGGAGGTCGTCACTAGCACCTG GGTGCTGGTGGGCGGAGTCCTTGCAGCTCTGGCCGCGTATTGCCTGACAACAGGCAGTGTGGTCATTGTG GGTAGGATTATCTTGTCCGGGAGGCCGGCTATTGTTCCCGACAGGGAGCTTCTCTACCAGGAGTTCGATG AAATGGAAGAGTGCGCCACGCACCTCCCTTACATTGAGCAGGGAATGCAGCTCGCCGAGCAGTTCAAGCA GAAAGCGCTCGGGTTACTGCAAACAGCCACCAAACAAGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAG TGGCGAGCCCTTGAGACATTCTGGGCGAAGCACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAG GCTTATCCACTCTGCCTGGGAACCCCGCAATAGCATCATTGATGGCATTCACAGCCTCTATCACCAGCCC GCTCACCACCCAAAGTACCCTCCTGTTTAACATCTTGGGGGGGTGGGTGGCTGCCCAACTCGCCCCCCCC AGCGCCGCTTCGGCTTTCGTGGGCGCCGGCATCGCCGGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGG TGCTTGTGGACATTCTGGCGGGTTATGGAGCAGGAGTGGCCGGCGCGCTCGTGGCCTTTAAGGTCATGAG CGGCGAGATGCCCTCTACCGAGGACCTGGTCAATCTACTTCCTGCCATCCTCTCTCCTGGCGCCCTGGTC GTCGGGGTCGTGTGTGCAGCAATACTGCGTCGGCACGTGGGTCCGGGAGAGGGGGCTGTGCAGTGGATGA ACCGGCTGATAGCGTTCGCCTCGCGGGGTAATCACGTTTCCCCCACGCACTATGTGCCTGAGAGCGACGC CGCAGCGCGTGTTACTCAGATCCTCTCCAGCCTTACCATCACTCAGCTGCTGAAAAGGCTCCACCAGTGG ATTAATGAGGACTGCTCCACACCGTGTTCCGGCTCGTGGCTAAGGGATGTTTGGGACTGGATATGCACGG TGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCAGCTACCGGGAGTCCCTTTTTTCTC GTGCCAACGCGGGTACAAGGGAGTCTGGCGGGGAGACGGCATCATGCAAACCACCTGCCCATGTGGAGCA CAGATCACCGGACATGTCAAAAACGGTTCCATGAGGATCGTCGGGCCTAAGACCTGCAGCAACACGTGGC ATGGAACATTCCCCATCAACGCATACACCACGGGCCCCTGCACACCCTCTCCAGCGCCAAACTATTCTAG GGCGCTGTGGCGGGTGGCCGCTGAGGAGTACGTGGAGGTCACGCGGGTGGGGGATTTCCACTACGTGACG GGCATGACCACTGACAACGTAAAGTGCCCATGCCAGGTTCCGGCTCCTGAATTCTTCTCGGAGGTGGACG GAGTGCGGTTGCACAGGTACGCTCCGGCGTGCAGGCCTCTCCTACGGGAGGAGGTTACATTCCAGGTCGG GCTCAACCAATACCTGGTTGGGTCACAGCTACCATGCGAGCCCGAACCGGATGTAGCAGTGCTCACTTCC ATGCTCACCGACCCCTCCCACATCACAGCAGAAACGGCTAAGCGTAGGTTGGCCAGGGGGTCTCCCCCCT CCTTGGCCAGCTCTTCAGCTAGCCAGTTGTCTGCGCCTTCCTTGAAGGCGACATGCACTACCCACCATGT CTCTCCGGACGCTGACCTCATCGAGGCCAACCTCCTGTGGCGGCAGGAGATGGGCGGGAACATCACCCGC GTGGAGTCGGAGAACAAGGTGGTAGTCCTGGACTCTTTCGACCCGCTTCGAGCGGAGGAGGATGAGAGGG AAGTATCCGTTCCGGCGGAGATCCTGCGGAAATCCAAGAAGTTCCCCGCAGCGATGCCCATCTGGGCGCG CCCGGATTACAACCCTCCACTGTTAGAGTCCTGGAAGGACCCGGACTACGTCCCTCCGGTGGTGCACGGG TGCCCGTTGCCACCTATCAAGGCCCCTCCAATACCACCTCCACGGAGAAAGAGGACGGTTGTCCTAACAG AGTCCTCCGTGTCTTCTGCCTTAGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCCGAATCATCGGCCGT CGACAGCGGCACGGCGACCGCCCTTCCTGACCAGGCCTCCGACGACGGTGACAAAGGATCCGACGTTGAG TCGTACTCCTCCATGCCCCCCCTTGAGGGGGAACCGGGGGACCCCGATCTCAGTGACGGGTCTTGGTCTA CCGTGAGCGAGGAAGCTAGTGAGGATGTCGTCTGCTGCTCAATGTCCTACACATGGACAGGCGCCTTGAT CACGCCATGCGCTGCGGAGGAAAGCAAGCTGCCCATCAACGCGTTGAGCAACTCTTTGCTGCGCCACCAT AACATGGTTTATGCCACAACATCTCGCAGCGCAGGCCTGCGGCAGAAGAAGGTCACCTTTGACAGACTGC AAGTCCTGGACGACCACTACCGGGACGTGCTCAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAA ACTCCTATCCGTAGAGGAAGCCTGCAAGCTGACGCCCCCACATTCGGCCAAATCCAAGTTTGGCTATGGG GCAAAGGACGTCCGGAACCTATCCAGCAAGGCCGTTAACCACATCCACTCCGTGTGGAAGGACTTGCTGG AAGACACTGTGACACCAATTGACACCACCATCATGGCAAAAAATGAGGTTTTCTGTGTCCAACCAGAGAA AGGAGGCCGTAAGCCAGCCCGCCTTATCGTATTCCCAGATCTGGGAGTCCGTGTATGCGAGAAGATGGCC CTCTATGATGTGGTCTCCACCCTTCCTCAGGTCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTG GGCAGCGAGTCGAGTTCCTGGTGAATACCTGGAAATCAAAGAAAAACCCCATGGGCTTTTCATATGACAC TCGCTGTTTCGACTCAACGGTCACCGAGAACGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGAC TTGGCCCCCGAAGCCAGACAGGCCATAAAATCGCTCACAGAGCGGCTTTATATCGGGGGTCCTCTGACTA ATTCAAAAGGGCAGAACTGCGGTTATCGCCGGTGCCGCGCGAGCGGCGTGCTGACGACTAGCTGCGGTAA CACCCTCACATGTTACTTGAAGGCCTCTGCAGCCTGTCGAGCTGCGAAGCTCCAGGACTGCACGATGCTC GTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACCCAAGAGGACGCGGCGAGCCTACGAG TCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCCGGGGACCCGCCCCAACCAGAATACGACTTGGA GCTGATAACATCATGTTCCTCCAATGTGTCGGTCGCCCACGATGCATCAGGCAAAAGGGTGTACTACCTC ACCCGTGATCCCACCACCCCCCTCGCACGGGCTGCGTGGGAAACAGCTAGACACACTCCAGTTAACTCCT GGCTAGGCAACATTATCATGTATGCGCCCACTTTGTGGGCAAGGATGATTCTGATGACTCACTTCTTCTC CATCCTTCTAGCACAGGAGCAACTTGAAAAAGCCCTGGACTGCCAGATCTACGGGGCCTGTTACTCCATT GAGCCACTTGACCTACCTCAGATCATTGAACGACTCCATGGCCTTAGCGCATTTTCACTCCATAGTTACT CTCCAGGTGAGATCAATAGGGTGGCTTCATGCCTCAGGAAACTTGGGGTACCACCCTTGCGAGTCTGGAG ACATCGGGCCAGGAGCGTCCGCGCTAGGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTAC CTCTTCAACTGGGCAGTGAAGACCAAACTCAAACTCACTCCAATCCCGGCTGCGTCCCAGCTGGACTTGT CCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGCTG GTTCATGCTGTGCCTACTCCTACTTTCTGTAGGGGTAGGCATCTACCTGCTCCCCAACCGATGAACGGGG AGCTAAACACTCCAGGCCAATAGGCCATTTCCTGTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTTCT TTTCCTTCTTTTTCCCTTTTTCTTTCTTCCTTCTTTAATGGTGGCTCCATCTTAGCCCTAGTCACGGCTA GCTGTGAAAGGTCCGTGAGCCGCATGACTGCAGAGAGTGCTGATACTGGCCTCTCTGCAGATCATGT (SEQ ID NO: 6688) gi|306286|gb|M96362.1|HPCUNKCDS Hepatitis C virus mRNA, complete cds TGCCAGCCCCCGATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGC AGAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCC ATAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCC GCTCAATGCCTGGAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGC CTTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCAC GAATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGATATTAAGTTCCCGGGC GGTGGTCAGATCGTTGGTGGAGTTTACTTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTA GGAAGACTTCCGAGCGGTCGCAACCTCGTGGAAGGCGACAGCCTATCCCCAAGGCTCGCCGGCCCGAGGG CAGGGCCTGGGCTCAGCCCGGGTACCCTTGGCCCCTCTATGGCAATGAGGGCTTGGGGTGGGCAGGATGG CTCCTGTCACCCCGCGGCTCCCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAAGTCGCGTAATTTGG GTAAGGTCATCGACACCCTCACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTCGTCGGCGCCCC CCTAGGGGGCGTTGCCAGGGCCCTGGCACATGGTGTCCGGGTGCTGGAGGACGGCGTGAACTATGCAACA GGGAATCTGCCCGGTTGCTCTTTCTCTATCTTCCTCTTGGCTCTGCTGTCTTGTTTGACCACCCCAGTTT CCGCTTATGAAGTGCGTAACGCGTCCGGGATGTACCATGTCACGAACGACTGCTCCAACTCAAGCATTGT GTATGAGGCAGCGGACATGATCATGCACACTCCCGGGTGCGTGCCCTGCGTTCGGGAGGACAACTCCTCC CGTTGCTGGGTGGCACTTACTCCCACGCTCGCGGCCAGGAATGCCAGCGTCCCCACTACGACATTGCGAC GCCATGTCGACTTGCTCGTTGGGGTAGCTGCTTTCTGTTCCGCTATGTACGTGGGGGACCTCTGCGGATC TGTTTTCCTTGTTTCCCAGCTGTTCACCTTTTCGCCTCGCCGGCATGAGACGGTACAGGACTGCAACTGC TCAATCTATCCCGGCCGCGTATCAGGTCACCGCATGGCCTGGGATATGATGATGAACTGGTCGCCTACAA CAGCCCTAGTGGTATCGCAGCTACTCCGGATCCCACAAGCTGTCGTGGACATGGTGACAGGGTCCCACTG GGGAATCCTGGCGGGCCTTGCCTACTATTCCATGGTGGGGAACTGGGCTAAGGTCTTAATTGCGATGCTA CTCTTTGCCGGCGTTGACGGAACCACCCACGTGACAGGGGGGGCGCAAGGTCGGGCCGCTAGCTCGCTAA CGTCCCTCTTTAGCCCTGGGCCGGTTCAGCACCTCCAGCTCATAAACACCAACGGCAGCTGGCATATCAA CAGGACCGCCCTGAGCTGCAATGACTCCCTCAACACTGGGTTTGTTGCCGCGCTGTTCTACAAATACAGG TTCAACGCGTCCGGGTGCCCGGAGCGCTTGGCCACGTGCCGCCCCATTGATACATTCGCGCAGGGGTGGG GTCCCATCACTTACACTGAGCCTCATGATTTGGATCAGAGGCCCTATTGCTGGCACTACGCGCCTCAACC GTGTGGTATTGTGCCCACGTTGCAGGTGTGTGGCCCAGTATACTGCTTCACCCCGAGTCCTGTTGCGGTG GGGACTACCGATCGTTTCGGTGCCCCTACATACAGATGGGGGGCAAATGAGACGGACGTGCTGCTCCTTA ACAACGCCGGGCCGCCGCAAGGCAACTGGTTCGGCTGTACATGGATGAATGGCACTGGGTTCACCAAGAC ATGTGGGGGCCCCCCGTGTAACATCGGGGGGGTCGGCAACAATACCTTGACCTGCCCCACGGACTGCTTC CGAAAGCACCCCGGGGCCACTTACACCAAATGCGGTTCGGGGCCTTGGTTAACACCCAGGTGCTTAGTCG ACTACCCGTACAGGCTCTGGCATTACCCCTGCACTGTCAACTTTACCATCTTTAAGGTTAGGATGTACGT GGGGGGCGCGGAGCACAGGCTCGACGCCGCATGCAACTGGACTCGGGGAGAGCGTTGTGACCTGGAGGAC AGGGATAGGTCAGAGCTTAGCCCGCTGCTGCTGTCTACAACAGAGTGGCAGGTACTGCCCTGTTCCTTCA CAACCCTACCGGCTCTGTCCACTGGTTTGATTCATCTCCATCAGAACATCGTGGACATACAATACCTGTA CGGTATAGGGTCGGCGGTTGTCTCCTTTGCGATCAAATGGGAGTATATTGTGCTGCTCTTCCTTCTTCTG GCGGACGCGCGCGTCTGCGCTTGCTTGTGGATGATGCTGCTGGTAGCGCAAGCCGAGGCCGCCTTAGAGA ACCTGGTGGTCCTCAATGCAGCGTCCGTGGCCGGAGCGCATGGCATTCTTTCCTTCATTGTGTTCTTCTG TGCTGCCTGGTACATCAAGGGCAGGCTGGTTCCCGGAGCGGCATACGCCCTCTATGGCGTATGGCCGCTG CTTCTGCTTCTGCTGGCGTTACCACCACGGGCGTACGCCATGGACCGGGAGATGGCCGCATCGTGCGGAG GCGCGGTTTTTGTAGGTCTGGTACTCTTGACCTTGTCACCACACTATAAAGTGTTCCTTGCCAGGTTCAT ATGGTGGCTACAATATCTCATCACCAGAACCGAAGCGCATCTGCAAGTGTGGGTCCCCCCTCTCAACGTT CGGGGGGGTCGCGATGCCATCATCCTCCTCACATGCGTGGTCCACCCAGAGCTAATCTTTGACATCACAA AATATTTGCTCGCCATATTCGGCCCGCTCATGGTGCTCCAGGCCGGCATAACTAGAGTGCCGTACTTCGT GCGCGCACAAGGGCTCATTCGTGCATGCATGTTGGCGCGGAAAGTCGTGGGGGGTCATTACGTCCAAATG GTCTTCATGAAGCTGGCCGCACTAGCAGGTACGTACGTTTATGACCATCTTACTCCACTGCGAGATTGGG CTCACACGGGCTTACGAGACCTTGCAGTGGCAGTAGAGCCCGTTGTCTTCTCTGACATGGAGACCAAAGT CATCACCTGGGGGGCAGACACCGCGGCGTGCGGGGACATCATCTTGGCCCTGCCTGCTTCCGCCCGAAGG GGGAAGGAGATACTTCTGGGACCGGCCGATAGTCTTGAAGGACAGGGGTGGCGACTCCTTGCGCCCATCA CGGCCTACTCCCAACAAACGCGAGGCCTGCTTGGTTGCATCATCACTAGCCTTACAGGCCGGGACAAGAA CCAGGTTGAGGGGGAGGTTCAAGTGGTTTCCACCGCAACACAATCTTTCCTGGCGACCTGCATCAATGGC GTGTGTTGGACTGTCTTCCACGGCGCCGGCTCAAAGACCCTAGCCGGCCCAAAGGGTCCAATCACCCAAA TGTACACCAATGTAGACCAGGACCTTGTTGGCTGGCCGGCACCTCCTGGGGCGCGTTCCCTGACACCATG CACTTGCGGCTCCTCGGACCTTTACCTGGTCACGAGACATGCTGATGTCATTCCGGTGCGCCGGCGGGGT GACGGTAGGGGGAGCCTACTCCCCCCCAGGCCTGTCTCCTACTTGAAGGGCTCCTCGGGTGGTCCACTGC TCTGCCCTTCGGGGCACGCTGTCGGCATACTTCCGGCTGCTGTATGCACCCGGGGGGTTGCCATGGCGGT GGAATTCATACCCGTTGAGTCTATGGAAACTACTATGCGGTCTCCGGTCTTCACGGACAATCCGTCTCCC CCGGCTGTACCGCAGACATTCCAAGTGGCCCACTTACACGCTCCCACCGGCAGCGGCAAGAGCACTAGGG TGCCGGCTGCATATGCAGCCCAAGGGTACAAGGTGCTCGTCCTAAATCCGTCCGTCGCCGCCACCTTGGG TTTTGGGGCGTATATGTCCAAGGCACATGGTATCGACCCCAACCTTAGAACTGGGGTAAGGACCATCACC ACAGGTGCCCCTATCACATACTCCACCTATGGCAAGTTCCTTGCCGACGGTGGCGGCTCCGGGGGCGCCT ATGACATCATAATGTGTGATGAGTGCCACTCAACTGACTCGACTACCATTTATGGCATCGGCACAGTCCT GGACCAAGCGGAGACGGCTGGAGCGCGGCTCGTGGTGCTCTCCACCGCTACGCCTCCGGGATCGGTCACC GTGCCACACCTCAATATCGAGGAGGTGGCCCTGTCTAATACTGGAGAGATCCCCTTCTACGGCAAAGCCA TTCCCATCGAGGCTATCAAGGGGGGAAGGCATCTCATTTTCTGCCATTCCAAGAAGAAGTGTGACGAACT CGCCGCAAAGCTGTCAGGCCTCGGACTCAATGCCGTAGCGTATTACCGGGGTCTTGACGTGTCCGTCATA CCGACCAGCGGAGACGTTGTTGTCGTGGCGACGGACGCTCTAATGACGGGCTTTACCGGCGACTTTGACT CAGTGATCGACTGTAATACGTGTGTCACCCAGACAGTCGATTTCAGCTTGGACCCCACCTTCACCATTGA GACGACGACCGTGCCCCAAGACGCAGTGTCGCGCTCGCAGAGGCGAGGCAGGACTGGTAGGGGCAGGGCT GGCATATACAGGTTTGTGACTCCAGGAGAACGGCCCTCGGGCATGTTCGATTCTTCGGTCCTGTGTGAGT GTTATGACGCGGGTTGTGCGTGGTACGAACTCACGCCCGCTGAGACCTCGGTTAGGTTGCGGGCGTACCT AAACACACCAGGGTTGCCCGTCTGCCAGGACCATCTGGAGTTCTCGGAGGGTGTCTTCACAGGCCTCACC CACATAGATGCCCACTTCTTATCCCAGACTAAACAGGCAGGAGAGAACTTCCCCTACTTGGTAGCATACC AGGCTACAGTGTGCGCCAGGGCTCAAGCCCCACCTCCATCGTGGGATGAAATGTGGAGGTGTCTCATACG GCTGAAACCTACGCTGCACGGGCCAACACCCCTGCTGTATAGGTTAGGAGCCGTCCAAAATGAGGTCACC CTCACACACCCCATAACCAAATTCATCATGACATGTATGTCGGCTGACCTGGAGGTCGTCACCAGCACCT GGGTGCTGGTAGGCGGAGTCCTCGCAGCTCTGGCCGCGTACTGCCTGACAACAGGCAGCGTGGTCATTGT GGGCAGGATCATCCTGTCCGGGAAGCCGGCTATCATCCCCGATAGGGAAGTTCTCTACCAGGAGTTCGAC GAGATGGAGGAGTGTGCCTCACACCTCCCTTACTTCGAACAGGGAATGCAGCTCGCCGAGCAATTCAAAC AGAAGGCGCTCGGGTTGCTGCAAACAGCCACCAAGCAGGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAA GTGGCGAGCCCTTGAGACCTTCTGGGCGAAGCACATGTGGAACTTCATTAGTGGGATACAGTACTTGGCA GGCTTGTCCACTCTGCCTGGGAACCCCGCAATACGATCACCGATGGCATTCACAGCCTCCATCACCAGCC CGCTCACCACCCAGCATACCCTCTTGTTTAACATCTTGGGGGGATGGGTGGCTGCCCAACTCGCCCCCCC CAGCGCTGCCTCAGCTTTCGTGGGCGCCGGCATCGCTGGAGCCGCTGTTGGCACGATAGGCCTTGGGAAG GTGCTTGTGGACATTCTGGCAGGTTATGGAGCAGGGGTGGCGGGCGCACTTGTGGCCTTTAAGATCATGA GCGGCGAGATGCCTTCAGCCGAGGACATGGTCAACTTACTCCCTGCCATCCTTTCTCCCGGTGCCCTGGT CGTCGGGATTGTGTGTGCAGCAATACTGCGTCGGCATGTGGGCCCAGGGGAAGGGGCTGTGCAGTGGATG AACCGGCTGATAGCGTTCGCCTCGCGGGGTAACCACGTCTCCCCCAGGCACTATGTGCCAGAGAGCGAGC CTGCAGCGCGTGTTACCCAGATCCTTTCCAGCCTCACCATCACTCAGCTGTTGAAGAGACTCCACCAGTG GATTAATGAGGACTGCTCTACGCCATGCTCCAGCTCGTGGCTAAGGGAGATTTGGGACTGGATCTGCACG GTGTTGACTGACTTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGATTACCGGGAGTCCCTTTTTTCT CATGCCAACGCGGGTATAAGGGAGTCTGGCGGGGGGACGGCATCATGCACACCACCTGCCCATGCGGAGC ACAGATCACCGGACACGTCAAAAACGGTTCCATGAGGATCGTTGGGCCTAAAACCTGCAGCAACACGTGG TACGGGACATTCCCCATCAACGCGTACACCACGGGCCCCTGCACACCCTCCCCGGCGCCAAACTATTCCA AGGCATTGTGGAGAGTGGCCGCTGAGGAGTACGTGGAGGTCACGCGGGTGGGAGATTTTCACTACGTGAC GGGCATGACCACTGACAACGTGAAGTGTCCATGCCAGGTTCCGGCCCCCGAATTCTTCACGGAGGTGGAT GGAGTGCGGTTGCACAGGTACGCTCCGGCGTGCAGACCTCTCCTACGGGAGGAGGTCGTATTCCAGGTCG GGCTCCACCAGTACCTGGTCGGGTCACAGCTCCCATGCGAGCCCGAACCGGATGTAGCAGTGCTCACTTC CATGCTCACTGACCCCTCCCACATTACAGCAGAGACGGCTAAGCGTAGGCTGGCCAGGGGGTCTCCCCCC TCCTTGGCCAGCTCTTCAGCTAGCCAGTTGTCTGCGCCTTCCTTGAAGGCGACATGCACTACCCATCATG ACTCCCCGGACGCTGACCTCATTGAGGCCAACCTCTTGTGGCGGCAAGAGATGGGCGGGAACATCACCCG CGTGGAGTCAGAGAATAAGGTGGTAATCCTGGACTCTTTCGACCCGCTCCGAGCGGAGGATGATGAGGGG GAAATATCCGTTCCGGCGGAGATCCTGCGGAAATCCAGGAAATTCCCCCCAGCGCTGCCCATATGGGCGC CGCCGGATTACAACCCTCCGCTGCTAGAGTCCTGGAAGGACCCGGACTACGTTCCTCCGGTGGTACACGG GTGCCCGTTGCCGCCCACCAAGGCCCCTCCAATACCACCTCCACGGAGGAAGAGGACGGTTGTCCTGACA GAATCCACCGTGTCTTCTGCCTTGGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCCGGATCGTCGGCCA TCGACAGCGGTACGGCGACCGCCCCTCCTGACCAAGCCTCCGGTGACGGCGACAGAGAGTCCGACGTTGA GTCGTTCTCCTCCATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGATCTCAGCGACGGATCTTGGTCC ACCGTGAGCGAGGAGGCTAGTGAGGACGTCGTCTGCTGTTCGATGTCCTACACATGGACAGGCGCCCTGA TCACGCCATGCGCTGCGGAGGAAAGCAAGTTGCCCATCAACCCGTTGAGCAATTCTTTGCTACGTCACCA CAACATGGTCTATGCTACAACATCCCGCAGCGCAGGCCTGCGGCAGAAGAAGGTCACCTTTGACAGACTG CAAGTCCTGGACGACCACTACCGGGACGTGCTTAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTA AACTTCTATCTGTAGAAGAAGCCTGCAAACTGACGCCCCCACATTCGGCCAAATCCAAATTTGGCTACGG GGCGAAGGACGTCCGGAGCCTATCCAGCAGGGCCGTTACCCACATCCGCTCCGTGTGGAAGGACCTGCTG GAAGACACTGAAACACCAATTAGCACTACCATCATGGCAAAAAATGAGGTTTTCTGTGTCCAACCAGAGA AGGGAGGCCGCAAGCCAGCTCGCCTTATCGTGTTCCCAGATCTGGGAGTTCGTGTATGCGAGAAGATGGC CCTTTATGACGTGGTCTCCACCCTTCCTCAGGCCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCT AAGCAGCGGGTCGAGTTCCTGGTGAATACCTGGAAATCAAAGAAATGCCCCATGGGCTTCTCATATGACA CCCGCTGTTTTGACTCAACGGTCACTGAGAATGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGA CTTGGCCCCCGAAGCCAAACTGGCCATAAAGTCGCTCACAGAGCGGCTCTATATCGGGGGTCCCCTGACT AATTCAAAAGGGCAGAACTGCGGTTACCGCCGGTGCCGCGCGAGCGGCGTGCTGACGACTAGCTGCGGTA ATACCCTCACATGTTACCTGAAAGCCACTGCGGCCTGTCGAGCTGCGAAGCTCCGGGACTGCACGATGCT CGTGAACGGAGACGACCTTGTCGTTATCTGTGAAAGCGCGGGAACCCAAGAGGATGCGGCGAGCCTACGA GTCTTCACGGAGGCTATGACTAGGTACTCTGCCCCCCCTGGGGACCCGCCTCAACCGGAATACGACTTGG AGTTGATAACATCATGTTCCTCCAATGTGTCGGTCGCACACGATGCATCTGGTAAAAGGGTGTACTACCT CACCCGTGACCCTACCACCCCCCTTGCACGGGCTGCGTGGGAGACAGCTAGACACACTCCAGTCAACTCC TGGCTAGGCAACATCATCATGTATGCGCCCACCTTATGGGCAAGGATGATTCTGATGACTCATTTCTTCT CCATCCTTCTAGCTCAGGAGCAACTTGAAAAAACCCTAGATTGTCAGATCTACGGGGCCTGTTACTCCAT TGAACCACTTGATCTACCTCAGATCATTGAGCGACTCCATGGTCTTAGCGCATTTTCACTCCATAGTTAC TCTCCAGGCGAGATCAATAGGGTGGCTTCATGCCTCAGAAAACTTGGGGTACCACCCTTGCGAGCCTGGA GACATCGGGCCAGAAGTGTCCGCGCTAAGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTA CCTCTTCAACTGGGCGGTGAGGACCAAGCTCAAACTCACTCCAATCCCAGCCGCGTCCCGGTTGGACTTG TCCGGCTGGTTCGTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGCT GGTTCATGTTGTGCCTACTCCTACTTTCCGTGGGGGTAGGCATCTACCTGCTCCCCAACCGATGAATGGG GAGCTAAACACTCCAGGCCAATAGGCCGTTTCTC (SEQ ID NO: 6689) gi|329739|gb|L02836.1|HPCCGENOM Hepatitis C China virus complete genome ATTGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCAGAAAGCGTCTA GCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCATAGTGGTCTGC GGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCGCTCAATGCCTG GAGATTTGGGCGTGCCCCCGCGAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCCTTGTGGTACTG CCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACGAATCCTAAACC TCAAAGAAAAACCAAACGTAACACCAACCGCCGCCCACAGGACGTCAAGTTCCCGGGCGGTGGTCAGATC GTTGGTGGAGTTTACCTGTTGCCGCGCAGGGGCCCCAGGTTGGGTGTGCGCGCGACTAGGAAGACTTCCG AGCGGTCGCAACCTCGTGGAAGGCGACAACCTATCCCCAAGGCTCGCCGACCCGAGGGCAGGACCTGGGC TCAGCCCGGGTATCCTTGGCCCCTCTATGGCAATGAGGGCTTTGGGTGGGCAGGATGGCTCCTGTCACCC CGCGGCTCCCGGCCTAGTTGGGGCCCCACGGACCCCCGGCGTAGGTCGCGTAATTTGGGTAAGGTCATCG ATACCCTCACATGCGGCTTCGCCGACCTCATGGGGTACATTCCGCTCGTCGGCGCCCCCTTGGGGGGCGC TGCCAGGGCCCTGGCACATGGTGTCCGGGTTCTGGAGGACGGCGTGAACTATGCAACAGGGAATTTGCCC GGTTGCTCTTTCTCTATCTTCCTTTTAGCCTTGCTATCCTGTTTGACCACCCCAGCTTCCGCTTACGAAG TGCGTAACGTGTCCGGGATATACCATGTCACGAACGACTGCTCCAACTCAAGCATTGTGTATGAGGCAGC GGACCTGATCATGCATACCCCTGGGTGCGTGCCCTGCGTTCGGGAAGGCAACTCCTCCCGTTGCTGGGTA GCGCTCACTCCCACGCTCGCGGCCAGGAACGCCACGATCCCCACTGCGACAGTACGACGGCATGTCGATC TGCTCGTTGGGGCGGCTGCTTTCTCTTCCGCCATGTACGTGGGGGATCTCTGCGGATCTGTTTTCCTTGT CTCTCAGCTGTTCACCTTCTCGCCTCGCCGGTATGAGACAATACAGGACTGCAATTGCTCAATCTATCCC GGCCACGTAACAGGTCACCGCATGGCTTGGGATATGATGATGAACTGGTCGCCTACAACAGCTCTAGTGG TGTCGCAGTTACTCCGGATCCCTCAAGCCGTCATGGACATGGTGGTGGGGGCCCACTGGGGAGTCCTGGC GGGCCTTGCCTACTATGCCATGGTGGGGAATTGGGCTAAGGTTTTGATTGTGATGCTACTCTTCGCCGGC GTTGATGGGGATACCTACGCGTCTGGGGGGGCGCAGGGCCGCTCCACCCTCGGGTTCACGTCCCTCTTTA CACCTGGGGCCTCTCAGAAGATCCAGCTTATAAATACCAATGGTAGCTGGCATATCAACAGGACTGCCCT GAACTGCAATGACTCCCTCAATACTGGGTTTCTTGCCGCGCTGTTCTATACACACAGGTTCAACGCGTCC GGATGCGCAGAGCGCATGGCCAGCTGCCGCCCCATTGATACATTCGATCAGGGCTGGGGCCCCATCACTT ATACTGAGCCTGATAGCTCGGACCAGAGGCCTTATTGCTGGCACTACGCGCCTCGAAAGTGCGGCATCGT ACCTGCGTCGGAGGTGTGCGGTCCAGTGTATTGTTTCACCCCAAGCCCTGTCGTCGTGGGGACGACCGAT CGTTTCGGTGTCCCCACATATAGCTGGGGGGAGAATGAGACAGACGTGCTGCTCCTCAACAACACGCGGC CGCCGCAAGGCAACTGGTTTGGCTGTACATGGATGAATGGCACTGGGTTCACCAAGACGTGCGGGGGGCC TCCGTGTAACATCGGGGGGGTCGGCAACAACACTTTGACTTGCCCCACGGATTGCTTTCGGAAGCACCCC GAGGCTACGTATACAAGGTGTGGTTCGGGGCCTTGGCTGACACCTAGGTGCTTAGTTGACTACCCATACA GGCTCTGGCACTACCCCTGCACTGTCAACTTTGCCATCTTCAAAGTTAGGATGTATGTGGGGGGCGTGGA GCACAGGCTCGATGCTGCATGCAACTGGACTCGAGGAGAGCGCTGTAACTTGGAGGACAGGGATAGATCA GAACTCAGCCCGCTGCTACTGTCTACAACAGAGTGGCAGATACTACCCTGCGCCTTCACCACCCTACCGG CTCTGTCCACTGGTTTAATCCATCTCCATCAGAACATCGTGGACGTGCAATACCTGTACGGTATAGGGTC AGCGGTTGCCTCCTTTGCAATTAAATGGGAGTATGTCTTGTTGCTTTTCCTTCTACTAGCAGACGCGCGC GTATGTGCCTGCTTGTGGATGATGCTGCTGATAGCCCAGGCCGAGGCCGCCTTAGAGAACCTGGTGGTCC TCAATGCGGCGTCCGTGGCCGACGCGCATGGCATCCTCTCCTTCCTTGTGTTCTTTTGTGCCGCCTGGTA CATTAAGGGCAGGCTGGTCCCCGGGGCAGCATACGCTTTCTACGGCGTGTGGCCGCTGCTCCTGCTCCTG CTGACATTACCACCACGAGCTTACGCCATGGACCGGGAGATGGCTGCATCGTGCGGAGGCGCGGTTTTTG TAGGTCTGGTATTCCTGACTTTGTCACCATACTACAAGGTGTTCCTCGCTAGGCTCATATGGTGGTTGCA ATACTTCCTCACCATAGCCGAGGCGCACCTGCAAGTGTGGATCCCCCCTCTCAACATTCGAGGGGGCCGC GATGCCATCATCCTCCTCACGTGTGCAATCCACCCAGAGTCAATCTTTGACATCACCAAACTCCTGCTCG CCACGCTCGGTCCGCTCCTGGTGCTTCAGGCTGGCATAACTAGAGTGCCGTACTTTGTGCGCGCTCATGG GCTCATTCGCGCGTGCATGCTATTGCGGAAAGTTGCTGGGGGTCATTATGTCCAAATGGCCTTCATGAAG CTGGGCGCACTGACAGGTACGTACGTCTATAACCATCTTACTCCGCTGCAGTATTGGCCACGCGCGGGTT TACGAGAACTCGCGGTGGCAGTAGAGCCCGTCATCTTCTCTGACATGGAGACCAAGATTATCACCTGGGG GGCAGACACTGCAGCGTGTGGAGACATCATCTTGGGTTTACCCGTCTCCGCCCGAAGGGGAAAGGAGATA CTCCTGGGGCCGGCCGATAGTCTTGAAGGGCAGGGGTGGCGACTCCTTGCGCCCATCACGGCCTACTCCC AACAGACGCGGGGCTTACTTGGTTGCATCATCACTAGCCTCACAGGCCGAGACAAGAACCAGGTCGAGGG GGAGGTTCAAGTGGTCTCCACCGCAACACAATCTTTCCTGGCGACCTGCATCAACGGTGTGTGTTGGACT GTCTATCATGGCGCCGGCTCAAAAACCTTAGCCGGCCCAAAGGGCCCAATCACCCAAATGTACACCAATG TAGACCAGGACCTCGTCGGCTGGCACCGGCCCCCCGGGGCGCGTTCCCTAACACCATGCACCTGCGGCAG CTCGGACCTTTACTTGGTCACGAGACATGCTGATGTCATTCCGGTGCGCCGTCGAGGCGACAGTAGGGGG AGTTTACTCTCCCCCAGGCCTGTCTCCTACCTGAAGGGCTCGTCGGGGGGCCCACTGCTCTGCCCCTTCG GGCACGTTGCAGGCATCTTCCGGGCTGCTGTGTGCACCCGGGGGGTTGCGAAGGCGGTGGATTTTATACC CGTTGAGACCATGGAAACTACCATGCGGTCCCCGGTCTTCACGGACAACTCATCCCCTCCTGCCGTACCG CAGACATTCCAAGTGGCCCATCTACACGCTCCCACTGGCAGCGGCAAAAGCACCAAGGTGCCGGCTGCAT ATGCAGCCCAAGGGTACAAGGTACTTGTCTTGAACCCGTCTGTTGCCGCCACTTTAGGTTTTGGGGCGTA TATGTCTAAGGCACATGGTGTCGACCCCAACATTAGAACCGGGGTAAGGACCATCACCACGGGCGCCCCC ATCACATACTCTACCTATGGCAAGTTCCTTGCTGATGGTGGTTGCTCTGGGGGTGCCTATGACATTATAA TATGTGATGAGTGCCATTCAACTGACTCGACTACCATCTTGGGCATCGGCACGGTCCTGGACCAAGCGGA GACGGCTGGAGCGCGGCTTGTCGTGCTCGCCACCGCTACGCCTCCGGGATCGGTCACCGTGCCACATCCA AACATCGAGGAGGTGGCCCTGTCCAATACTGGAGAGATCCCCTTCTATGGTAAAGCCATCCCCATCGAAG CCATCAGGGGGGGAAGGCATCTCATTTTCTGCCACTCCAAGAAGAAGTGTGACGAGCTTGCTGCAAAGCT ATCATCGCTCGGGCTCAACGCTGTGGCGTACTACCGGGGGCTTGATGTGTCCGTCATACCATCTAGCGGA GACGTCGTTGTCGTGGCAACGGACGCTCTAATGACGGGCTTTACGGGCGACTTTGACTCAGTGATCGACT GTAACACATGTGTTACCCAAACAGTCGATTTCAGCTTGGACCCCACCTTCACCATCGAGACAACGACCGT GCCCCAAGACGCGGTGTCGCGCTCGCAGCGGCGAGGTAGGACTGGCAGGGGTAGGGAAGGCATCTACAGG TTTGTTACTCCAGGAGAACGGCCCTCGGGCATGTTCGACTCCTCAGTCCTGTGTGAGTGCTATGACGCGG GCTGTGCTTGGTACGAGCTCACGCCGGCTGAGACCACGGTTAGGTTGCGGGCTTACCTAAATACACCAGG GTTGCCCGTCTGCCAGGACCATCTGGAGTTCTGGGAGGGCGTCTTCACAGGTCTCACCCATATAGACGCT CACTTTCTGTCCCAGACCAAGCAAGCAGGAGACAACTTCCCCTACCTGGTAGCATACCAAGCTACAGTGT GTGCCAAGGCTCAGGCCCCACCTCCATCGTGGGATCAAATGTGGAAGTGCCTCACACGGCTAAAGCCTAC GCTGCAGGGACCAACACCCCTGCTGTATAGGCTAGGAGCCGTCCAAAATGAGGTCACCCTCACACACCCC ATAACTAAATACATCATGACATGCATGTCGGCTGACCTGGAGGTCGTCACCAGCACCTGGGTGCTGGTGG GCGGAGTCCTTGCAGCTCTGGCCGCGTATTGCCTGACAACGGGCAGCGTGGTCATTGTGGGTAGGATTGT CTTGTCCGGAAGTCCGGCTATTGTTCCTGACAGGGAAGTTCTTTACCAAGACTTCGACGAGATGGAAGAG TGTGCCTCACACCTCCCTTACATCGAACAGGGAATGCAGCTCGCCGAGCAGTTCAAGCAGAAGGCGCTCG GGTTGCTGCAAACAGCCACCAAGCAAGCGGAGGCTGCTGCTCCCGTGGTGGAGTCCAAGTGGCGAGCCCT CGAGACATTTTGGGAAAAACACATGTGGAATTTCATCAGCGGGATACAGTACTTAGCAGGCTTATCCACT CTGCCTGGGAACCCCGCAATGGCATCACTGATGGCATTCACAGCTTCTATCACCAGCCCGCTCACTACCC AACACACCCTCCTGTTTAACATCTTGGGTGGATGGGTGGCTGCCCAACTCGCTCCCCCCAGCGCCGCTTC GGCCTTTGTGGGCGCCGGCATTGCCGGTGCGGCTGTTGGCAGCATAGGCCTTGGGAAGGTGCTTGTGGAC ATCCTGGCGGGTTATGGGGCGGGGGTGGCTGGCGCACTCGTGGCCTTTAAGGTCATGAGTGGCGAAATGC CCTCCACTGAGGACCTGGTTAATTTACTCCCTGCCATCCTCTCTCCTGGTGCCCTAGTCGTCGGGGTCGT GTGCGCAGCAATACTGCGCCGACACGTGGGCCCGGGAGAGGGGGCTGTGCAGTGGATGAACCGGCTGATA GCGTTCGCTTCGCGGGGTAACCATGTCTCCCCCACGCACTATGTGCCTGAAAGTGACGCCGCAGCGCGTG TTACCCAGATCCTCTCCAGCCTTACCATCACTCAGCTGCTGAAAAGACTTCACCAGTGGATTAATGAGGA CTGTTCCACACCATGCTCCGGCTCGTGGCTAAGGGATGTTTGGGATTGGATATGCACGGTGTTGACCGAT TTCAAGACCTGGCTCCAGTCCAAGCTCCTGCCGCGGTTGCCCGGAGTCCCTTTCCTCTCATGCCAACGCG GGTACAAGGGAGTCTGGCGGGGGGACGGTATTATGCAAACCACCTGTCCATGTGGAGCACAGATTACTGG ACATGTCAAAAACGGTTCCATGAGAATCGTTGGGCCTAAGACTTGTAGCAACACGTGGCATGGAACATTC CCCATCAACGCGTACACCACGGGCCCCTGCACACCCTCCCCGGCGCCGAACTATTCCAGGGCGCTGTGGC GGGTGGCTCCTGAGGAGTACGTGGAGGTTACGCGGGTGGGGGATTTCCACTACGTGACGGGCATGACCAC CGACAACGTGAAATGCCCATGCCAAGTCCCGGCCCCTGAATTCTTCACGGAGGTGGATGGAGTACGGCTG CACAGGTACGCTCCGGCGTGCAAACCTCTCCTACGGGAGGAGGTCGTGTTCCAGGTCGGGCTCAACCAAT ACCTGGTTGGATCACAGCTCCCATGCGAGCCCGAGCCGGACGTAACAGTGCTCACTTCCATGCTTACCGA CCCCTCCCACATCACAGCAGAGACGGCCAAGCGTAGGCTGGCCAGGGGGTCTCCCCCCTCCTTGGCCAGC TCTTCAGCTAGCCAATTGTCTGCGCCTTCTTTGAAGGCGACATGTACTACCCATCATGACTCCCCGGACG CCGACCTCATTGAGGCCAACCTCCTGTGGCGGCAGGAGATGGGCGGAAACATCACCCGTGTGGAGTCAGA AAATAAGGTAGTGATCCTGGACTCTTTCGACCCGCTTCGGGCGGAGGAGGACGAGAGGGAAGTATCCGTT GCGGCGGAGATCCTGCGGAAATCCAGGAAGTTCCCCTCAGCGCTGCCCATATGGGCACGCCCAGACTACA ACCCTCCACTGCTAGAGTCCTGGAAGGACCCAGATTATGTCCCTCCGGTGGTACACGGGTGCCCGTTGCC GCCTACCACGGCCCCTCCAGTACCACCTCCACGGAGAAAAAGGACGGTCGTCCTAACAGAGTCATCCGTG TCTTCTGCCTTGGCGGAGCTCGCTACTAAGACCTTCGGCAGCTCTGAATCGTCGGCCGTCGACAGCGGCA CGGCGACTGCCCCTCCTGACGAGGCCTCCGGCGGCGGCGACAAAGGATCCGACGTTGAGTCGTACTCCTC CATGCCCCCCCTTGAGGGAGAGCCGGGGGACCCCGACCTCAGCGACGGGTCCTGGTCTACCGTGAGTGAG GAGGCCAGTGAGGACGTCGTCTGCTGCTCAATGTCCTATACATGGACAGGCGCCTTGATCACGCCATGTG CTGCGGAGGAGAGCAAGCTGCCCATCAACCCGCTGAGCAACTCCTTGCTGCGTCACCACAACATGGTCTA TGCTACAACATCCCGCAGTGCAAGCCTACGGCAGAAGAAGGTCGCTTTTGACAGAATGCAAGTCCTGGAC GACCACTACCGGGACGTGCTCAAGGAGATGAAGGCGAAGGCGTCCACAGTTAAGGCTAAACTCCTATCCA TAGAAGAGGCCTGCAAGCTGACGCCCCCACATTCAGCCAAATCCAAATTTGGCTATGGGGCAAAAGACGT CCGGAACCTATCCAGCAAGGCCGTTAACCACATCCGCTCCGTGTGGAAGGACTTGTTGGAAGACAATGAG ACACCAATCAATACCACCATCATGGCAAAAAATGAGGTTTTCTGCGTCCAACCAGAGAAAGGAGGCCGTA AGCCAGCTCGCCTTATCGTATTCCCAGACTTGGGAGTCCGTGTGTGCGAGAAGATGGCCCTTTATGACGT GGTCTCCACCCTTCCTCAGCCCGTGATGGGCTCCTCATACGGATTCCAGTACTCTCCTGGGCAGCGGGTC GAATTCCTGCTAAATGCCTGGAAATCAAAGGAAAACCCTATGGGCTTCTCATATGACACCCGCTGTTTTG ACTCAACGGTCACTCAGAACGACATCCGTGTTGAGGAGTCAATTTACCAATGTTGTGACTTGGCCCCCGA GGCCAGACGGGCCATAAAGTCGCTCACAGAGCGGCTCTATATCGGGGGTCCCCTGACTAATTCAAAAGGG CAGAACTGCGGTTATCGCCGGTGCCGCGCAAGTGGCGTGCTGACGACCAGCTGCGGTAATACCCTTACAT GTTACTTGAAGGCCTCTGCGGCCTGTCGAGCTGCGAAGCTGCAGGACTGCACGATGCTCGTGAACGGAGA CGACCTTGTCGTTATCTGTGAAAGCGCGGGAACTCAAGAGGATGCGGCGAGCCTACGAGTCTTCACGGAG GCTATGACTAGGTACTCTGCCCCCCCTGGGGACCTGCCCCAACCAGAATACGACTTGGAGCTAATAACAT CATGCTCCTCCAATGTGTCAGTCGCCCACGATGCATCTGGCAAAAGGGTGTACTACCTCACCCGTGACCC CACCATCCCCCTCGCGCGGGCTGCGTGGGAGACAGCTAGACACACTCCAGTCAACTCCTGGCTAGGCAAC ATCATCATGTATGCGCCCACTCTATGGGCAAGGATGATTCTGATGACTCACTTCTTCTCCATCCTTCTAG CTCAGGAGCAACTTGAGAAAGCCCTGGATTGCCAAATCTACGGGGCCTACTACTCCATTGAGCCACTTGA CCTACCTCAGATCATTGAACGACTCCATGGCCTTAGCGCATTTTCACTCCATAGTTACTCTCCAGGTGAG ATCAATAGGGTGGCGTCATGTCTCAGGAAACTTGGGGTACCACCCTTGCGAGTCTGGAGACATCGGGCCA GAAGCGTCCGCGCTAAGCTACTGTCCCAGGGGGGGAGGGCCGCCACTTGTGGCAAGTACCTCTTCAACTG GGCAGTAAAGACCAAGCTTAAACTCACTCCAATCCCGGCTGCGTCCCGGTTGGACTTGTCCGGCTGGTTC GTTGCTGGTTACAGCGGGGGAGACATATATCACAGCCTGTCTCGTGCCCGACCCCGTTGGTTCATGTTGT GCCTACTCCTACTTTCTGTAGGGGTAGGCATCTACCTGCTCCCCAACCGATGAACGGGGAGATAAACACT CCAGGCCAATAGGCCATCCC (SEQ ID NO: 6690) gi|15422182|gb|AY051292.1| Hepatitis C virus from India polyprotein mRNA, complete cds GCCAGCCCCCTGATGGGGGCGACACTCCACCATAGATCACTCCCCTGTGAGGAACTACTGTCTTCACGCA GAAAGCGTCTAGCCATGGCGTTAGTATGAGTGTCGTGCAGCCTCCAGGACCCCCCCTCCCGGGAGAGCCA TAGTGGTCTGCGGAACCGGTGAGTACACCGGAATTGCCAGGACGACCGGGTCCTTTCTTGGATCAACCCG CTCAATGCCTGGAGATTTGGGCGTGCCCCCGCAAGACTGCTAGCCGAGTAGTGTTGGGTCGCGAAAGGCC TTGTGGTACTGCCTGATAGGGTGCTTGCGAGTGCCCCGGGAGGTCTCGTAGACCGTGCACCATGAGCACG AATCCTAAACCTCAAAGAAAAACCAAACGTAACACCAACCGACGCCCACAGAACGTTAAGTTCCCGGGTG GCGGCCAGATCGTTGGCGGAGTTTGCTTGTTGCCGCGCAGGGGTCCCAGAGTGGGTGTGCGCGCGACGAG GAAGACTTCCGAGCGGTCACAACCTCGCGGAAGGCGTCAGCCTATTCCCAAGGCCCGCCGACCCGAGGGC AGGTCCTGGGCGCAGCCCGGGTACCCTTGGCCCCTCTATGGCAACGAGGGCTGTGGGTGGGCAGGATGGC TCTTGTCCCCCCGCGGCTCCCGGCCTAGTCGGGGCCCCTCTGACCCCCGGCGCAGGTCACGCAATTTGGG TAAGGTCATCGATACCCTCACGTGTGGCTTCGCCGACCTCATGGGGTACATCCCGCTCGTCGGTGCTCCT CTAGGGGGCGCTGCTAGGGCTCTGGCACATGGTGTTAGGGTTCTAGAAGACGGCGTAAATTACGCAACAG GGAACCTTCCTGGTTGCTCTTTTTCTATCTTCTTGCTTGCTCTTCTCTCCTGCTTGACAGTCCCTGCTTC GGCCGTCGAAGTGCGCAACTCTTCGGGGATCTACCATGTCACCAATGATTGCCCCAATGCGTCTGTTGTG TACGAGACAGATAGCTTGATCATACATCTGCCCGGGTGTGTGCCCTGCGTACGCGAGGGCAACGCTTCGA GGTGCTGGGTCTCCCTTAGTCCTACTGTTGCCGCTAAGGATCCGGGCGTCCCCGTCAACGAGATTCGGCG TCACGTCGACCTGATTGTCGGGGCCGCTGCATTCTGTTCGGCTATGTATGTAGGGGACTTATGCGGTTCC ATCTTCCTCGTTGGCCAGCTTTTCACCCTCTCCCCTAGGCGCCACTGGACAACACAAGACTGTAATTGCT CCATCTACCCAGGACATGTGACAGGCCATCGAATGGCTTGGGACATGATGATGAATTGGTCACCTACTGG CGCTTTGGTGGTAGCGCAGCTACTCCGGATCCCACAAGCCGTCTTGGATATGATAGCCGGTGCCCACTGG GGTGTCCTAGCGGGCCCGGCATACTACTCCATGGTGGGGAACTGGGCTAAGGTTTTGGTTGTGCTACTGC TCTTCGCTGGCGTCGATGCAACCACCCAAGTCACAGGTGGCACCGCGGGCCGTAATGCATATAGATTGGC TAGCCTCTTCTCCACCGGCCCCAGCCAAAATATCCAGCTCATAAACTCCAATGGCAGCTGGCACATTAAC AGGACTGCCCTGAATTGCAATGACAGCCTGCACACCGGCTGGGTAGCAGCGCTGTTCTACTCCCACAAGT TCAACTCTTCGGGGCGTCCTGAGAGGATGGCTAGTTGTCGGCCTCTTACCGCCTTCGACCAAGGGTGGGG GCCCATCACTTACGGGGGGAAAGCTAGTAACGACCAGCGGCCGTATTGCTGGCACTATGCCCCACGCCCG TGCGGTATCGTGCCGGCGAAAGAGGTTTGCGGGCCTGTATACTGTTTCACACCCAGTCCCGTGGTAGTGG GGACGACGGACAAGTACGGCGTTCCTACCTACACATGGGGCGAGAATGAGACGGATGTACTGCTCCTTAA CAACTCTAGGCCGCCAATAGGGAATTGGTTCGGGTGTACGTGGATGAATTCCACTGGTTTCACCAAGACG TGCGGGGCTCCTGCCTGTAACGTCGGCGGGAGCGAGACCAACACCCTGTCGTGCCCCACAGATTGCTTCC GCAGACATCCGGACGCAACATACGCTAAGTGCGGCTCTGGCCCTTGGCTTAACCCTCGATGCATGGTGGA CTACCCTTACAGGCTCTGGCACTATCCCTGCACAGTCAATTACACCATATTCAAGATCAGGATGTTCGTG GGCGGGATTGAGCACAGGCTCACCGCCGCGTGCAACTGGACGCGGGGAGAGCGCTGCGACTTGGACGACA GGGATCGTGCCGAGTTGAGCCCGCTGTTGCTGTCCACCACGCAATGGCAGGTCCTCCCCTGCTCATTCAC AACGCTGCCCGCCCTGTCAACTGGCCTAATACATCTCCACCAGAACATCGTGGACGTGCAGTACCTCTAC GGGTTGAGCTCGGTAGTTACATCCTGGGCCATAAGGTGGGAGTATGTCGTGCTCCTTTTCTTGCTGTTAG CAGATGCCCGCATTTGTGCCTGCCTTTGGATGATGCTTCTCATATCCCAGGTAGAGGCGGCGCTGGAGAA CCTGATAGTCCTCAACGCTGCTTCCCTGGCTGGGACACACGGCATCGTCCCTTTCTTCATCTTTTTTTGT GCAGCCTGGTATCTGAAAGGCAAGTGGGCCCCTGGACTCGTCTACTCCGTCTACGGAATGTGGCCGCTGC TCCTGCTTCTCCTGGCGTTGCCCCAACGGGCGTACGCCTTGGATCAGGAGTTGGCCGCGTCGTGTGGGGC CGTGGTCTTCATCAGCCTAGCGGTACTTACCCTGTCGCCGTACTACAAACAGTACATGGCCCGCGGCATC TGGTGGCTGCAGTACATGCTGACCAGAGCGGAGGCGCTCCTGCACGTCTGGGTCCCCTCGCTCAACGCCC GGGGAGGGCGTGATGGTGCCATACTGCTCATGTGTGTGCTCCACCCGCACTTGCTCTTTGACATCACCAA AATCATGCTGGCCATTCTCGGGCCCCTGTGGATCTTGCAGGCCAGTCTGCTCAGGGTGCCGTACTTCGTG CGCGCCCACGGTCTCATTAGGCTCTGCATGCTGGTGCGCAAAACAGCGGGCGGTCACTATGTGCAGATGG CTCTGTTGAAGCTGGGGGCACTTACTGGCACTTACATTTACAACCACCTTTCCCCACTCCAAGACTGGGC TCATGGCAGCTTGCGTGATCTAGCGGTGGCCACCGAGCCCGTCATCTTCTCCCGGATGGAGATCAAGACT ATCACCTGGGGGGCAGACACCGCGGCCTGTGGAGACATCATCAACGGGCTGCCTGTTTCTGCTCGGAGGG GGAGAGAGGTGTTGTTGGGACCAGCCGATGCCCTGACTGACAAGGGATGGAGGCTTTTAGCCCCCATCAC AGCTTACGCCCAACAGACACGAGGTCTCTTGGGCTGTATTGTCACCAGCCTCACCGGTCGGGACAAAAAT CAAGTGGAGGGGGAAATCCAGATTGTGTCTACCGCAACCCAGACGTTCTTGGCCACTTGCATCAACGGAG CTTGCTGGACTGTTTATCATGGGGCCGGATCGAGGACCATCGCTTCGGCGTCGGGTCCTGTGGTCCGGAT GTACACCAATGTGGACCAGGATTTGGTGGGCTGGCCAGCGCCTCAGGGAGCGCGCTCCCTGACGCCGTGC ACGTGCGGTGCCTCGGATCTGTACTTGGTCACGAGGCACGCGGATGTCATCCCAGTGCGGCGTCGAGGCG ATAACAGGGGAAGCTTGCTTTCTCCCCGGCCCATCTCATACCTAAAAGGATCCTCGGGAGGCCCTCTGCT CTGCCCCATGGGACATGTCGCGGGCATTTTTAGGGCCGCGGTGTGCACCCGTGGGGTTGCAAAGGCGGTC GACTTTGTGCCCGTTGAGTCCTTAGAGACCACCATGAGGTCCCCAGTGTTTACTGACAATTCCAGCCCTC CAACAGTGCCCCAGAGTTACCAGGTGGCACATCTACATGCACCCACTGGGAGTGGCAAGAGCACGAAGGT GCCGGCCGCTTACGCAGCTCAAGGGTACAAGGTACTTGTGCTGAACCCGTCTGTTGCTGCCACCTTAGGG TTCGGTGCTTATATGTCAAAGGCCCATGGGATTGACCCAAACGTCAGGACCGGCGTGAGGACCATTACCA CAGGCTCCCCCATCACCTACTCCACCTACGGGAAATTTTTGGCTGATGGCGGATGCCCAGGAGGTGCGTA CGACATCATAATATGTGACGAATGTCACTCAGTGGACGCCACCTCGATTCTGGGCATAGGGACCGTCTTG GACCAAGCGGAGACGGCGGGGGTTAGGCTCACTGTCCTTGCCACCGCTACACCACCTGGCTTGGTCACCG TGCCACATTCCAACATCGAGGAAGTTGCACTGTCCGCTGACGGGGAGAAACCATTTTATGGTAAGGCCAT CCCCCTAAACTACATCAAGGGGGGGAGGCATCTCATTTTCTGTCATTCCAAGAAGAAGTGCGACGAGCTC GCTGCAAAGCTGGTCGGTCTGGGCGTCAACGCGGTGGCCTTTTACCGTGGCCTCGACGTATCTGTCATTC CAACTACAGGAGACGTCGTTGTTGTAGCGACCGACGCCTTGATGACTGGCTTCACCGGCGATTTCGACTC TGTGATAGACTGCAACACCTGTGTCGTCCAGACAGTCGACTTCAGCCTAGACCCTATATTCTCTATTGAG ACTTCCACCGTGCCCCAGGACGCCGTGTCCCGCTCCCAACGGAGGGGTAGGACCGGTCGAGGGAAGCATG GTATTTACAGATATGTGTCACCCGGGGAGCGGCCGTCTGGCATGTTCGACTCCGTGGTCCTCTGTGAGTG CTATGACGCGGGTTGTGCTTGGTACGAGCTTACACCCGCCGAGACCACAGTCAGGCTACGGGCATACCTT AACACCCCAGGATTGCCCGTGTGCCAGGACCACTTGGAGTTCTGGGAGAGTGTCTTCACCGGCCTCACCC ACATAGATGCCCACTTCCTGTCCCAGACGAAACAGAGTGGGGAGAACTTCCCCTACCTAGTCGCATACCA AGCCACCGTGTGCGCTAGAGCTAGAGCTCCTCCCCCGTCATGGGACCAAATGTGGAAGTGCCTGATACGG CTCAAGCCCACCCTCACTGGGGCTACCCCATTACTATACAGACTGGGTAGTGTACAGAATGAGATCACCT TAACACACCCAATCACCCAATACATCATGGCTTGCATGTCGGCGGACCTGGAGGTCGTCACTAGCACGTG GGTGTTGGTGGGCGGCGTCCTAGCCGCTTTGGCCGCTTACTGCCTGTCCACAGGCAGCGTGGTCATAGTG GGCAGGATAATCCTAGGTGGGAAGCCGGCAGTCATACCTGACAGGGAGGTTCTCTACCGAGAGTTTGATG AGATGGAGGAGTGCGCCGCCCACGTCCCCTACCTCGAGCAGGGGATGCATTTGGCTGGACAGTTCAAGCA GAAAGCTCTCGGGTTGCTCCAGACAGCATCCAAGCAAGCGGAGACGATCACTCCCACTGTCCGCACCAAC TGGCAGAAACTCGAGTCCTTCTGGGCTAAGCACATGTGGAACTTCGTTAGCGGGATACAATACCTGGCGG GCCTGTCAACGCTGCCCGGGAACCCCGCTATAGCGTCGCTGATGTCGTTTACGGCCGCGGTGACGAGTCC ACTAACCACCCAGCAAACCCTCTTCTTTAACATCTTAGGGGGGTGGGTGGCGGCCCAGCTTGCTTCCCCA GCTGCCGCTACTGCTTTTGTCGGTGCTGGTATTACTGGCGCCGTTGTTGGCAGTGTGGGCCTAGGGAAGG TCCTAGTGGACATTATTGCTGGCTACGGGGCTGGTGTGGCGGGGGCCCTCGTGGCTTTCAAAATCATGAG CGGGGAGACCCCCACCACCGAGGATCTAGTCAACCTTCTGCCTGCCATCCTATCGCCAGGAGCTCTCGTT GTCGGCGTGGTGTGCGCAGCAATACTACGCCGGCACGTGGGCCCTGGCGAGGGCGCCGTGCAGTGGATGA ACCGGCTGATAGCGTTTGCTTCTCGGGGTAACCACGTCTCCCCTACACACTACGTGCCGGAGAGCGACGC GTCGGCTCGTGTCACACAAATTCTCACCAGCCTCACTGTTACTCAGCTTCTGAAAAGGCTCCACGTGTGG ATAAGCTCGGATTGCATCGCCCCGTGTGCTAGTTCTTGGCTTAAAGATGTCTGGGACTGGATATGCGAGG TGCTGAGCGACTTCAAGAATTGGCTGAAGGCCAAACTTGTACCACAACTGCCCGGGATCCCATTCGTATC CTGCCAACGCGGGTACCGTGGGGTCTGGCGGGGCGAGGGCATCGTGCACACTCGTTGCCCGTGTGGGGCC AATATAACTGGACATGTCAAGAACGGTTCGATGAGAATCGTCGGGCCTAAGACTTGCAGCAACACCTGGC GTGGGTCGTTCCCCATTAACGCTTACACTACAGGCCCGTGCACGCCCTCCCCGGCGCCGAACTATACGTT CGCGCTATGGAGGGTGTCTGCAGAGGAGTATGTGGAGGTAAGGCGGCTGGGGGACTTCCATTACGTCACG GGGGTGACCACTGATAAACTCAAGTGTCCATGCCAGGTCCCCTCACCCGAGTTCTTCACAGAGGTGGACG GGGTGCGCCTGCATAGGTACGCCCCCCCCTGCAAACCCCTGCTGCGAGAAGAGGTGACGTTTAGCATCGG GCTCAATGAATACTTGGTGGGGTCCCAGTTGCCCTGCGAGCCCGAGCCAGACGTAGCTGTACTGACATCA ATGCTTACAGACCCCTCCCACATCACTGCAGAGACGGCAGCGCGTAGGCTGAAGCGGGGGTCTCCCCCCT CCCTGGCCAGCTCTTCCGCCAGCCAGCTGTCCGCGCCGTCACTGAAGGCAACATGCACCACTCACCACGA CTCTCCAGACGCTGACCTCATAGAAGCCAACCTCCTGTGGAGACAGGAGATGGGGGGGAACATCACTAGG GTGGAGTCGGAGAACAAGATTGTCGTTCTGGATTCTTTCGACCCGCTCGTAGCGGAGGAGGATGATCGGG AGATCTCTATTCCAGCTGAGATTCTGCGGAAGTTCAAGCAGTTTCCTCCCGCTATGCCCATATGGGCACG GCCAGATTATAATCCTCCCCTTGTGGAACCGTGGAAGCGCCCGGACTATGAGCCACCCTTAGTCCACGGG TGCCCCCTACCACCTCCCAAGCCAACTCCGGTGCCGCCACCCCGGAGAAAGAGGACGGTGGTGCTGGACG AGTCTACAGTATCATCTGCTCTGGCTGAGCTTGCCACTAAGACCTTCGGCAGCTCTACAACCTCAGGCGT GACAAGTGGTGAAGCGACTGAATCGTCCCCGGCGCCCTCCTGCGGCGGTGAGCTGGACTCCGAAGCTGAA TCTTACTCCTCCATGCCCCCTCTCGAGGGGGAGCCGGGGGACCCCGATCTCAGCGACGGGTCTTGGTCTA CCGTGAGCAGTGATGGTGGCACGGAAGACGTTGTGTGCTGCTCGATGTCTTACTCGTGGACGGGCGCTTT AATCACGCCCTGTGCCTCAGAGGAAGCCAAGCTCCCTATCAACGCATTGAGCAACTCGCTGCTGCGCCAC CACAACTTGGTGTATTCCACCACCTCTCGCAGCGCTGGCCAGAGACAGAAAAAAGTCACATTTGACAGAG TGCAAGTCCTGGACGACCATTACCGGGACGTGCTCAAGGAGGCTAAGGCCAAGGCATCCACGGTGAAGGC TAGACTGCTATCCGTTGAGGAAGCGTGTAGCCTGACGCCCCCACACTCCGCCAGATCAAAATTTGGCTAT GGGGCGAAGGATGTCCGAAGCCATTCCAGTAAGGCTATACGCCACATCAACTCCGTGTGGCAGGACCTTC TGGAGGACAATACAACACCCATAGACACTACCATCATGGCAAAGAATGAGGTCTTCTGTGTGAAGCCCGA AAAGGGGGGCCGCAAGCCCGCTCGTCTTATCGTGTACCCCGACCTGGGAGTGCGCGTATGCGAGAAGAGG GCTTTGTATGACGTAGTCAAACAGCTCCCCATTGCCGTGATGGGAGCCTCCTACGGGTTCCAGTACTCAC CAGCGCAGCGGGTCGACTTCCTGCTTAAAGCGTGGAAATCTAAGAAAGTCCCCATGGGGTTTTCCTATGA CACCCGTTGCTTTGACTCAACAGTCACTGAGGCTGATATCCGTACGGAGGAAGACCTCTACCAATCTTGT GACCTGGCCCCTGAGGCTCGCATAGCCATAAGGTCCCTCACAGAGAGGCTTTACATCGGGGGCCCACTCA CCAATTCTAAGGGACAAAACTGCGGCTATCGGCGATGCCGCGCAAGCGGCGTGCTGACCACTAGCTGCGG TAACACCATAACCTGCTTCCTCAAAGCCAGTGCAGCCTGTCGAGCTGCGAAGCTCCAGGACTGCACCATG CTCGTGTGCGGCGACGACCTCGTCGTTATCTGTGAGAGCGCCGGTGTCCAGGAGGACGCTGCGAGCCTGA GAGCCTTCACGGAGGCTATGACCAGGTACTCCGCCCCCCCGGGAGACCCGCCTCAACCAGAATACGACTT GGAGCTTATAACATCCTGCTCCTCCAATGTGTCGGTCGCGCGCGACGGCGCTGGCAAAAGGGTCTATTAT CTGACCCGTGACCCTGAGACTCCCCTCGCGCGTGCCGCTTGGGAGACAGCAAGACACACTCCAGTGAACT CCTGGCTAGGCAACATCATCATGTTTGCCCCCACTCTGTGGGTACGGATGGTCCTCATGACCCATTTTTT CTCCATACTCATAGCTCAGGAGCACCTTGGAAAGGCTCTAGATTGTGAAATCTATGGAGCCGTACACTCC GTCCAACCGTTGGACTTACCTGAAATCATCCAAAGACTCCACAGCCTCAGCGCGTTTTCGCTCCACAGTT ACTCTCCAGGTGAAATCAATAGGGTGGCTGCATGCCTCAGGAAGCTTGGGGTTCCGCCCTTGCGAGCTTG GAGACACCGGGCCCGGAGCGTTCGCGCCACACTCCTATCCCAGGGGGGGAAAGCCGCTATATGCGGTAAG TACCTCTTCAACTGGGCGGTGAAAACCAAACTCAAACTCACTCCATTACCGTCCATGTCTCAGTTGGACT TGTCCAACTGGTTCACGGGCGGTTACAGCGGGGGAGACATTTATCACAGCGTGTCTCATGCCCGGCCCCG TTTGTTCCTCTGGTGCCTACTCCTACTTTCAGTAGGGGTAGGCATCTATCTCCTTCCCAACCGATAGACG GNTGGGCAACCACTCCGGGTCTTTAGGCCCTATTTAAACACTCCAGGCCTTTAGGCCCCGT (SEQ ID NO: 6691) gi|23510419|ref|NM_000043.31| Homo sapiens tumor necrosis factor receptor superfamily, member 6 (TNFRSF6), transcript variant 1, mRNA CCTACCCGCGCGCAGGCCAAGTTGCTGAATCAATGGAGCCCTCCCCAACCCGGGCGTTCCCCAGCGAGGC TTCCTTCCCATCCTCCTGACCACCGGGGCTTTTCGTGAGCTCGTCTCTGATCTCGCGCAAGAGTGACACA CAGGTGTTCAAAGACGCTTCTGGGGAGTGAGGGAAGCGGTTTACGAGTGACTTGGCTGGAGCCTCAGGGG CGGGCACTGGCACGGAACACACCCTGAGGCCAGCCCTGGCTGCCCAGGCGGAGCTGCCTCTTCTCCCGCG GGTTGGTGGACCCGCTCAGTACGGAGTTGGGGAAGCTCTTTCACTTCGGAGGATTGCTCAACAACCATGC TGGGCATCTGGACCCTCCTACCTCTGGTTCTTACGTCTGTTGCTAGATTATCGTCCAAAAGTGTTAATGC CCAAGTGACTGACATCAACTCCAAGGGATTGGAATTGAGGAAGACTGTTACTACAGTTGAGACTCAGAAC TTGGAAGGCCTGCATCATGATGGCCAATTCTGCCATAAGCCCTGTCCTCCAGGTGAAAGGAAAGCTAGGG ACTGCACAGTCAATGGGGATGAACCAGACTGCGTGCCCTGCCAAGAAGGGAAGGAGTACACAGACAAAGC CCATTTTTCTTCCAAATGCAGAAGATGTAGATTGTGTGATGAAGGACATGGCTTAGAAGTGGAAATAAAC TGCACCCGGACCCAGAATACCAAGTGCAGATGTAAACCAAACTTTTTTTGTAACTCTACTGTATGTGAAC ACTGTGACCCTTGCACCAAATGTGAACATGGAATCATCAAGGAATGCACACTCACCAGCAACACCAAGTG CAAAGAGGAAGGATCCAGATCTAACTTGGGGTGGCTTTGTCTTCTTCTTTTGCCAATTCCACTAATTGTT TGGGTGAAGAGAAAGGAAGTACAGAAAACATGCAGAAAGCACAGAAAGGAAAACCAAGGTTCTCATGAAT CTCCAACCTTAAATCCTGAAACAGTGGCAATAAATTTATCTGATGTTGACTTGAGTAAATATATCACCAC TATTGCTGGAGTCATGACACTAAGTCAAGTTAAAGGCTTTGTTCGAAAGAATGGTGTCAATGAAGCCAAA ATAGATGAGATCAAGAATGACAATGTCCAAGACACAGCAGAACAGAAAGTTCAACTGCTTCGTAATTGGC ATCAACTTCATGGAAAGAAAGAAGCGTATGACACATTGATTAAAGATCTCAAAAAAGCCAATCTTTGTAC TCTTGCAGAGAAAATTCAGACTATCATCCTCAAGGACATTACTAGTGACTCAGAAAATTCAAACTTCAGA AATGAAATCCAAAGCTTGGTCTAGAGTGAAAAACAACAAATTCAGTTCTGAGTATATGCAATTAGTGTTT GAAAAGATTCTTAATAGCTGGCTGTAAATACTGCTTGGTTTTTTACTGGGTACATTTTATCATTTATTAG CGCTGAAGAGCCAACATATTTGTAGATTTTTAATATCTCATGATTCTGCCTCCAAGGATGTTTAAAATCT AGTTGGGAAAACAAACTTCATCAAGAGTAAATGCAGTGGCATGCTAAGTACCCAAATAGGAGTGTATGCA GAGGATGAAAGATTAAGATTATGCTCTGGCATCTAACATATGATTCTGTAGTATGAATGTAATCAGTGTA TGTTAGTACAAATGTCTATCCACAGGCTAACCCCACTCTATGAATCAATAGAAGAAGCTATGACCTTTTG CTGAAATATCAGTTACTGAACAGGCAGGCCACTTTGCCTCTAAATTACCTCTGATAATTCTAGAGATTTT ACCATATTTCTAAACTTTGTTTATAACTCTGAGAAGATCATATTTATGTAAAGTATATGTATTTGAGTGC AGAATTTAAATAAGGCTCTACCTCAAAGACCTTTGCACAGTTTATTGGTGTCATATTATACAATATTTCA ATTGTGAATTCACATAGAAAACATTAAATTATAATGTTTGACTATTATATATGTGTATGCATTTTACTGG CTCAAAACTACCTACTTCTTTCTCAGGCATCAAAAGCATTTTGAGCAGGAGAGTATTACTAGAGCTTTGC CACCTCTCCATTTTTGCCTTGGTGCTCATCTTAATGGCCTAATGCACCCCCAAACATGGAAATATCACCA AAAAATACTTAATAGTCCACCAAAAGGCAAGACTGCCCTTAGAAATTCTAGCCTGGTTTGGAGATACTAA CTGCTCTCAGAGAAAGTAGCTTTGTGACATGTCATGAACCCATGTTTGCAATCAAAGATGATAAAATAGA TTCTTATTTTTCCCCCACCCCCGAAAATGTTCAATAATGTCCCATGTAAAACCTGCTACAAATGGCAGCT TATACATAGCAATGGTAAAATCATCATCTGGATTTAGGAATTGCTCTTGTCATACCCCCAAGTTTCTAAG ATTTAAGATTCTCCTTACTACTATCCTACGTTTAAATATCTTTGAAAGTTTGTATTAAATGTGAATTTTA AGAAATAATATTTATATTTCTGTAAATGTAAACTGTGAAGATAGTTATAAACTGAAGCAGATACCTGGAA CCACCTAAAGAACTTCCATTTATGGAGGATTTTTTTGCCCCTTGTGTTTGGAATTATAAAATATAGGTAA AAGTACGTAATTAAATAATGTTTTTGGTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA AAAAAAAAAAAAAAAAAAAAAAAA (SEQ ID NO: 6692) gi|35910|emb|X12387.1|HSRCYP3 Human mRNA for cytochrome P-450 (cyp3 locus) GAATTCCCAAAGAGCAACACAGAGCTGAAAGGAAGACTCAGAGGAGAGAGATAAGTAAGGAAAGTAGTGA TGGCTCTCATCCCAGACTTGGCCATGGAAACCTGGCTTCTCCTGGCTGTCAGCCTGGTGCTCCTCTATCT ATATGGAACCCATTCACATGGACTTTTTAAGAAGCTTGGAATTCCAGGGCCCACACCTCTGCCTTTTTTG GGAAATATTTTGTCCTACCATAAGGGCTTTTGTATGTTTGACATGGAATGTCATAAAAAGTATGGAAAAG TGTGGGGCTTTTATGATGGTCAACAGCCTGTGCTGGCTATCACAGATCCTGACATGATCAAAACAGTGCT AGTGAAAGAATGTTATTCTGTCTTCACAAACCGGAGGCCTTTTGGTCCAGTGGGATTTATGAAAAGTGCC ATCTCTATAGCTGAGGATGAAGAATGGAAGAGATTACGATCATTGCTGTCTCCAACCTTCACCAGTGGAA AACTCAAGGAGATGGTCCCTATCATTGCCCAGTATGGAGATGTGTTGGTGAGAAATCTGAGGCGGGAAGC AGAGACAGGCAAGCCTGTCACCTTGAAAGACGTCTTTGGGGCCTACAGCATGGATGTGATCACTAGCACA TCATTTGGAGTGAACATCGACTCTCTCAACAATCCACAAGACCCCTTTGTGGAAAACACCAAGAAGCTTT TAAGATTTGATTTTTTGGATCCATTCTTTCTCTCAATAACAGTCTTTCCATTCCTCATCCCAATTCTTGA AGTATTAAATATCTGTGTGTTTCCAAGAGAAGTTACAAATTTTTTAAGAAAATCTGTAAAAAGGATGAAA GAAAGTCGCCTCGAAGATACACAAAAGCACCGAGTGGATTTCCTTCAGCTGATGATTGACTCTCAGAATT CAAAAGAAACTGAGTCCCACAAAGCTCTGTCCGATCTGGAGCTCGTGGCCCAATCAATTATCTTTATTTT TGCTGGCTATGAAACCACGAGCAGTGTTCTCTCCTTCATTATGTATGAACTGGCCACTCACCCTGATGTC CAGCAGAAACTGCAGGAGGAAATTGATGCAGTTTTACCCAATAAGGCACCACCCACCTATGATACTGTGC TACAGATGGAGTATCTTGACATGGTGGTGAATGAAACGCTCAGATTATTCCCAATTGCTATGAGACTTGA GAGGGTCTGCAAAAAAGATGTTGAGATCAATGGGATGTTCATTCCCAAAGGGTGGGTGGTGATGATTCCA AGCTATGCTCTTCACCGTGACCCAAAGTACTGGACAGAGCCTGAGAAGTTCCTCCCTGAAAGATTCAGCA AGAAGAACAAGGACAACATAGATCCTTACATATACACACCCTTTGGAAGTGGACCCAGAAACTGCATTGG CATGAGGTTTGCTCTCATGAACATGAAACTTGCTCTAATCAGAGTCCTTCAGAACTTCTCCTTCAAACCT TGTAAAGAAACACAGATCCCCCTGAAATTAAGCTTAGGAGGACTTCTTCAACCAGAAAAACCCGTTGTTC TAAAGGTTGAGTCAAGGGATGGCACCGTAAGTGGAGCCTGAATTTTCCTAAGGACTTCTGCTTTGCTCTT CAAGAAATCTGTGCCTGAGAACACCAGAGACCTCAAATTACTTTGTGAATAGAACTCTGAAATGAAGATG GGCTTCATCCAATGGACTGCATAAATAACCGGGGATTCTGTACATGCATTGAGCTCTCTCATTGTCTGTG TAGAGTGTTATACTTGGGAATATAAAGGAGGTGACCAAATCAGTGTGAGGAGGTAGATTTGGCTCCTCTG CTTCTCACGGGACTATTTCCACCACCCCCAGTTAGCACCATTAACTCCTCCTGAGCTCTGATAAGAGAAT CAACATTTCTCAATAATTTCCTCCACAAATTATTAATGAAAATAAGAATTATTTTGATGGCTCTAACAAT GACATTTATATCACATGTTTTCTCTGGAGTATTCTATAGTTTTATGTTAAATCAATAAAGACCACTTTAC AAAAGTATTATCAGATGCTTTCCTGCACATTAAGGAGAATCTATAGAACTGAATGAGAACCAACAAGTAA ATATTTTTGGTCATTGTAATCACTGTTGGCGTGGGGCCTTTGTCAGAACTAGAATTTGATTATTAACATA GGTGAAAGTTAATCCACTGTGACTTTGCCCATTGTTTAGAAAGAATATTCATAGTTTAATTATGCCTTTT TTGATCAGGCACATGGCTCACGCCTGTAATCCTAGCAGTTTGGGAGGCTGAGCCGGGTGGATCGCCTGAG GTCAGGAGTTCAAGACAAGCCTGGCCTACATGGTGAAACCCCATCTCTACTAAAAATACACAAATTAGCT AGGCATGGTGGACTCGCCTGTAATCTCACTACACAGGAGGCTGAGGCAGGAGAATCACTTGAACCTGGGA GGCGGATGTTGAAGTGAGCTGAGATTGCACCACTGCACTCCAGTCTGGGTGAGAGTGAGACTCAGTCTTA AAAAAATATGCCTTTTTGAAGCACGTACATTTTGTAACAAAGAACTGAAGCTCTTATTATATTATTAGTT TTGATTTAATGTTTTCAGCCCATCTCCTTTCATATTTCTGGGAGACAGAAAACATGTTTCCCTACACCTC TTGCTTCCATCCTCAACACCCAACTGTCTCGATGCAATGAACACTTAATAAAAAACAGTCGATTGGTCAA AAAAAAAAAAAAAAAAAAAAAAAGAATTC (SEQ ID NO: 6693) gi|339549|gb|M19154.1|HUMTGEB2A Human transforming growth factor-beta-2 mRNA, complete cds GCCCCTCCCGTCAGTTCGCCAGCTGCCAGCCCCGGGACCTTTTCATCTCTTCCCTTTTGGCCGGAGGAGC CGAGTTCAGATCCGCCACTCCGCACCCGAGACTGACACACTGAACTCCACTTCCTCCTCTTAAATTTATT TCTACTTAATAGCCACTCGTCTCTTTTTTTCCCCATCTCATTGCTCCAAGAATTTTTTTCTTCTTACTCG CCAAAGTCAGGGTTCCCTCTGCCCGTCCCGTATTAATATTTCCACTTTTGGAACTACTGGCCTTTTCTTT TTAAAGGAATTCAAGCAGGATACGTTTTTCTGTTGGGCATTGACTAGATTGTTTGCAAAAGTTTCGCATC AAAAACAACAACAACAAAAAACCAAACAACTCTCCTTGATCTATACTTTGAGAATTGTTGATTTCTTTTT TTTATTCTGACTTTTAAAAACAACTTTTTTTTCCACTTTTTTAAAAAATGCACTACTGTGTGCTGAGCGC TTTTCTGATCCTGCATCTGGTCACGGTCGCGCTCAGCCTGTCTACCTGCAGCACACTCGATATGGACCAG TTCATGCGCAAGAGGATCGAGGCGATCCGCGGGCAGATCCTGAGCAAGCTGAAGCTCACCAGTCCCCCAG AAGACTATCCTGAGCCCGAGGAAGTCCCCCCGGAGGTGATTTCCATCTACAACAGCACCAGGGACTTGCT CCAGGAGAAGGCGAGCCGGAGGGCGGCCGCCTGCGAGCGCGAGAGGAGCGACGAAGAGTACTACGCCAAG GAGGTTTACAAAATAGACATGCCGCCCTTCTTCCCCTCCGAAACTGTCTGCCCAGTTGTTACAACACCCT CTGGCTCAGTGGGCAGCTTGTGCTCCAGACAGTCCCAGGTGCTCTGTGGGTACCTTGATGCCATCCCGCC CACTTTCTACAGACCCTACTTCAGAATTGTTCGATTTGACGTCTCAGCAATGGAGAAGAATGCTTCCAAT TTGGTGAAAGCAGAGTTCAGAGTCTTTCGTTTGCAGAACCCAAAAGCCAGAGTGCCTGAACAACGGATTG AGCTATATCAGATTCTCAAGTCCAAAGATTTAACATCTCCAACCCAGCGCTACATCGACAGCAAAGTTGT GAAAACAAGAGCAGAAGGCGAATGGCTCTCCTTCGATGTAACTGATGCTGTTCATGAATGGCTTCACCAT AAAGACAGGAACCTGGGATTTAAAATAAGCTTACACTGTCCCTGCTGCACTTTTGTACCATCTAATAATT ACATCATCCCAAATAAAAGTGAAGAACTAGAAGCAAGATTTGCAGGTATTGATGGCACCTCCACATATAC CAGTGGTGATCAGAAAACTATAAAGTCCACTAGGAAAAAAAACAGTGGGAAGACCCCACATCTCCTGCTA ATGTTATTGCCCTCCTACAGACTTGAGTCACAACAGACCAACCGGCGGAAGAAGCGTGCTTTGGATGCGG CCTATTGCTTTAGAAATGTGCAGGATAATTGCTGCCTACGTCCACTTTACATTGATTTCAAGAGGGATCT AGGGTGGAAATGGATACACGAACCCAAAGGGTACAATGCCAACTTCTGTGCTGGAGCATGCCCGTATTTA TGGAGTTCAGACACTCAGCACAGCAGGGTCCTGAGCTTATATAATACCATAAATCCAGAAGCATCTGCTT CTCCTTGCTGCGTGTCCCAAGATTTAGAACCTCTAACCATTCTCTACTACATTGGCAAAACACCCAAGAT TGAACAGCTTTCTAATATGATTGTAAAGTCTTGCAAATGCAGCTAAAATTCTTGGAAAAGTGGCAAGACC AAAATGACAATGATGATGATAATGATGATGACGACGACAACGATGATGCTTGTAACAAGAAAACATAAGA GAGCCTTGGTTCATCAGTGTTAAAAAATTTTTGAAAAGGCGGTACTAGTTCAGACACTTTGGAAGTTTGT GTTCTGTTTGTTAAAACTGGCATCTGACACAAAAAAAGTTGAAGGCCTTATTCTACATTTCACCTACTTT GTAAGTGAGAGAGACAAGAAGCAAATTTTTTTTAAAGAAAAAAATAAACACTGGAAGAATTTATTAGTGT TAATTATGTGAACAACGACAACAACAACAACAACAACAAACAGGAAAATCCCATTAAGTGGAGTTGCTGT ACGTACCGTTCCTATCCCGCGCCTCACTTGATTTTTCTGTATTGCTATGCAATAGGCACCCTTCCCATTC TTACTCTTAGAGTTAACAGTGAGTTATTTATTGTGTGTTACTATATAATGAACGTTTCATTGCCCTTGGA AAATAAAACAGGTGTATAAAGTGGAGACCAAATACTTTGCCAGAAACTCATGGATGGCTTAAGGAACTTG AACTCAAACGAGCCAGAAAAAAAGAGGTCATATTAATGGGATGAAAACCCAAGTGAGTTATTATATGACC GAGAAAGTCTGCATTAAGATAAAGACCCTGAAAACACATGTTATGTATCAGCTGCCTAAGGAAGCTTCTT GTAAGGTCCAAAAACTAAAAAGACTGTTAATAAAAGAAACTTTCAGTCAG (SEQ ID NO: 6694) gi|186624|gb|J04111.1|HUMJUNA Human c-jun proto oncogene (JUN), complete cds, clone hCJ-1 CCCGGGGAGGGGACCGGGGAACAGAGGGCCGAGAGGCGTGCGGCAGGGGGGAGGGTAGGAGAAAGAAGGG CCCGACTGTAGGAGGGCAGCGGAGCATTACCTCATCCCGTGAGCCTCCGCGGGCCCAGAGAAGAATCTTC TAGGGTGGAGTCTCCATGGTGACGGGCGGGCCCGCCCCCCTGAGAGCGACGCGAGCCAATGGGAAGGCCT TGGGGTGACATCATGGGCTATTTTTAGGGGTTGACTGGTAGCAGATAAGTGTTGAGCTCGGGCTGGATAA GGGCTCAGAGTTGCACTGAGTGTGGCTGAAGCAGCGAGGCGGGAGTGGAGGTGCGCGGAGTCAGGCAGAC AGACAGACACAGCCAGCCAGCCAGGTCGGCAGTATAGTCCGAACTGCAAATCTTATTTTCTTTTCACCTT CTCTCTAACTGCCCAGAGCTAGCGCCTGTGGCTCCCGGGCTGGTGGTTCGGGAGTGTCCAGAGAGCCTTG TCTCCAGCCGGCCCCGGGAGGAGAGCCCTGCTGCCCAGGCGCTGTTGACAGCGGCGGAAAGCAGCGGTAC CCCACGCGCCCGCCGGGGGACGTCGGCGAGCGGCTGCAGCAGCAAAGAACTTTCCCGGCGGGGAGGACCG GAGACAAGTGGCAGAGTCCCGGAGCGAACTTTTGCAAGCCTTTCCTGCGTCTTAGGCTTCTCCACGGCGG TAAAGACCAGAAGGCGGCGGAGAGCCACGCAAGAGAAGAAGGACGTGCGCTCAGCTTCGCTCGCACCGGT TGTTGAACTTGGGCGAGCGCGAGCCGCGGCTGCCGGGCGCCCCCTCCCCCTAGCAGCGGAGGAGGGGACA AGTCGTCGGAGTCCGGGCGGCCAAGACCCGCCGCCGGCCGGCCACTGCAGGGTCCGCACTGATCCGCTCC GCGGGGAGAGCCGCTGCTCTGGGAAGTGAGTTCGCCTGCGGACTCCGAGGAACCGCTGCGCCCGAAGAGC GCTCAGTGAGTGACCGCGACTTTTCAAAGCCGGGTAGCGCGCGCGAGTCGACAAGTAAGAGTGCGGGAGG CATCTTAATTAACCCTGCGCTCCCTGGAGCGAGCTGGTGAGGAGGGCGCAGCGGGGACGACAGCCAGCGG GTGCGTGCGCTCTTAGAGAAACTTTCCCTGTCAAAGGCTCCGGGGGGCGCGGGTGTCCCCCGCTTGCCAG AGCCCTGTTGCGGCCCCGAAACTTGTGCGCGCACGCCAAACTAACCTCACGTGAAGTGACGGACTGTTCT ATGACTGCAAAGATGGAAACGACCTTCTATGACGATGCCCTCAACGCCTCGTTCCTCCCGTCCGAGAGCG GACCTTATGGCTACAGTAACCCCAAGATCCTGAAACAGAGCATGACCCTGAACCTGGCCGACCCAGTGGG GAGCCTGAAGCCGCACCTCCGCGCCAAGAACTCGGACCTCCTCACCTCGCCCGACGTGGGGCTGCTCAAG CTGGCGTCGCCCGAGCTGGAGCGCCTGATAATCCAGTCCAGCAACGGGCACATCACCACCACGCCGACCC CCACCCAGTTCCTGTGCCCCAAGAACGTGACAGATGAGCAGGAGGGGTTCGCCGAGGGCTTCGTGCGCGC CCTGGCCGAACTGCACAGCCAGAACACGCTGCCCAGCGTCACGTCGGCGGCGCAGCCGGTCAACGGGGCA GGCATGGTGGCTCCCGCGGTAGCCTCGGTGGCAGGGGGCAGCGGCAGCGGCGGCTTCAGCGCCAGCCTGC ACAGCGAGCCGCCGGTCTACGCAAACCTCAGCAACTTCAACCCAGGCGCGCTGAGCAGCGGCGGCGGGGC GCCCTCCTACGGCGCGGCCGGCCTGGCCTTTCCCGCGCAACCCCAGCAGCAGCAGCAGCCGCCGCACCAC CTGCCCCAGCAGATGCCCGTGCAGCACCCGCGGCTGCAGGCCCTGAAGGAGGAGCCTCAGACAGTGCCCG AGATGCCCGGCGAGACACCGCCCCTGTCCCCCATCGACATGGAGTCCCAGGAGCGGATCAAGGCGGAGAG GAAGCGCATGAGGAACCGCATCGCTGCCTCCAAGTGCCGAAAAAGGAAGCTGGAGAGAATCGCCCGGCTG GAGGAAAAAGTGAAAACCTTGAAAGCTCAGAACTCGGAGCTGGCGTCCACGGCCAACATGCTCAGGGAAC AGGTGGCACAGCTTAAACAGAAAGTCATGAACCACGTTAACAGTGGGTGCCAACTCATGCTAACGCAGCA GTTGCAAACATTTTGAAGAGAGACCGTCGGGGGCTGAGGGGCAACGAAGAAAAAAAATAACACAGAGAGA CAGACTTGAGAACTTGACAAGTTGCGACGGAGAGAAAAAAGAAGTGTCCGAGAACTAAAGCCAAGGGTAT CCAAGTTGGACTGGGTTCGGTCTGACGGCGCCCCCAGTGTGCACGAGTGGGAAGGACTTGGTCGCGCCCT CCCTTGGCGTGGAGCCAGGGAGCGGCCGCCTGCGGGCTGCCCCGCTTTGCGGACGGGCTGTCCCCGCGCG AACGGAACGTTGGACTTTCGTTAACATTGACCAAGAACTGCATGGACCTAACATTCGATCTCATTCAGTA TTAAAGGGGGGAGGGGGAGGGGGTTACAAACTGCAATAGAGACTGTAGATTGCTTCTGTAGTACTCCTTA AGAACACAAAGCGGGGGGAGGGTTGGGGAGGGGCGGCAGGAGGGAGGTTTGTGAGAGCGAGGCTGAGCCT ACAGATGAACTCTTTCTGGCCTGCTTTCGTTAACTGTGTATGTACATATATATATTTTTTAATTTGATTA AAGCTGATTACTGTCAATAAACAGCTTCATGCCTTTGTAAGTTATTTCTTGTTTGTTTGTTTGGGTATCC TGCCCAGTGTTGTTTGTAAATAAGAGATTTGGAGCACTCTGAGTTTACCATTTGTAATAAAGTATATAAT TTTTTTATGTTTTGTTTCTGAAAATTCCAGAAAGGATATTTAAGAAAATACAATAAACTATTGGAAAGTA CTCCCCTAACCTCTTTTCTGCATCATCTGTAGATCCTAGTCTATCTAGGTGGAGTTGAAAGAGTTAAGAA TGCTCGATAAAATCACTCTCAGTGCTTCTTACTATTAAGCAGTAAAAACTGTTCTCTATTAGACTTAGAA ATAAATGTACCTGATGTACCTGATGCTATGTCAGGCTTCATACTCCACGCTCCCCCAGCGTATCTATATG GAATTGCTTACCAAAGGCTAGTGCGATGTTTCAGGAGGCTGGAGGAAGGGGGGTTGCAGTGGAGAGGGAC AGCCCACTGAGAAGTCAAACATTTCAAAGTTTGGATTGCATCAAGTGGCATGTGCTGTGACCATTTATAA TGTTAGAAATTTTACAATAGGTGCTTATTCTCAAAGCAGGAATTGGTGGCAGATTTTACAAAAGATGTAT CCTTCCAATTTGGAATCTTCTCTTTGACAATTCCTAGATAAAAAGATGGCCTTTGTCTTATGAATATTTA TAACAGCATTCTGTCACAATAAATGTATTCAAATACCAATAACAGATCTTGAATTGCTTCCCTTTACTAC TTTTTTGTTCCCAAGTTATATACTGAAGTTTTTATTTTTAGTTGCTGAGGTT (SEQ ID NO: 6695) gi|179982|gb|M57729.1|HUMCCC5 Human complement component C5 mRNA, complete cds CTACCTCCAACCATGGGCCTTTTGGGAATACTTTGTTTTTTAATCTTCCTGGGGAAAACCTGGGGACAGG AGCAAACATATGTCATTTCAGCACCAAAAATATTCCGTGTTGGAGCATCTGAAAATATTGTGATTCAAGT TTATGGATACACTGAAGCATTTGATGCAACAATCTCTATTAAAAGTTATCCTGATAAAAAATTTAGTTAC TCCTCAGGCCATGTTCATTTATCCTCAGAGAATAAATTCCAAAACTCTGCAATCTTAACAATACAACCAA AACAATTGCCTGGAGGACAAAACCCAGTTTCTTATGTGTATTTGGAAGTTGTATCAAAGCATTTTTCAAA ATCAAAAAGAATGCCAATAACCTATGACAATGGATTTCTCTTCATTCATACAGACAAACCTGTTTATACT CCAGACCAGTCAGTAAAAGTTAGAGTTTATTCGTTGAATGACGACTTGAAGCCAGCCAAAAGAGAAACTG TCTTAACCTTCATAGATCCTGAAGGATCAGAAGTTGACATGGTAGAAGAAATTGATCATATTGGAATTAT CTCTTTTCCTGACTTCAAGATTCCGTCTAATCCTAGATATGGTATGTGGACGATCAAGGCTAAATATAAA GAGGACTTTTCAACAACTGGAACCGCATATTTTGAAGTTAAAGAATATGTCTTGCCACATTTTTCTGTCT CAATCGAGCCAGAATATAATTTCATTGGTTACAAGAACTTTAAGAATTTTGAAATTACTATAAAAGCAAG ATATTTTTATAATAAAGTAGTCACTGAGGCTGACGTTTATATCACATTTGGAATAAGAGAAGACTTAAAA GATGATCAAAAAGAAATGATGCAAACAGCAATGCAAAACACAATGTTGATAAATGGAATTGCTCAAGTCA CATTTGATTCTGAAACAGCAGTCAAAGAACTGTCATACTACAGTTTAGAAGATTTAAACAACAAGTACCT TTATATTGCTGTAACAGTCATAGAGTCTACAGGTGGATTTTCTGAAGAGGCAGAAATACCTGGCATCAAA TATGTCCTCTCTCCCTACAAACTGAATTTGGTTGCTACTCCTCTTTTCCTGAAGCCTGGGATTCCATATC CCATCAAGGTGCAGGTTAAAGATTCGCTTGACCAGTTGGTAGGAGGAGTCCCAGTAATACTGAATGCACA AACAATTGATGTAAACCAAGAGACATCTGACTTGGATCCAAGCAAAAGTGTAACACGTGTTGATGATGGA GTAGCTTCCTTTGTGCTTAATCTCCCATCTGGAGTGACGGTGCTGGAGTTTAATGTCAAAACTGATGCTC CAGATCTTCCAGAAGAAAATCAGGCCAGGGAAGGTTACCGAGCAATAGCATACTCATCTCTCAGCCAAAG TTACCTTTATATTGATTGGACTGATAACCATAAGGCTTTGCTAGTGGGAGAACATCTGAATATTATTGTT ACCCCCAAAAGCCCATATATTGACAAAATAACTCACTATAATTACTTGATTTTATCCAAGGGCAAAATTA TCCATTTTGGCACGAGGGAGAAATTTTCAGATGCATCTTATCAAAGTATAAACATTCCAGTAACACAGAA CATGGTTCCTTCATCCCGACTTCTGGTCTATTATATCGTCACAGGAGAACAGACAGCAGAATTAGTGTCT GATTCAGTCTGGTTAAATATTGAAGAAAAATGTGGCAACCAGCTCCAGGTTCATCTGTCTCCTGATGCAG ATGCATATTCTCCAGGCCAAACTGTGTCTCTTAATATGGCAACTGGAATGGATTCCTGGGTGGCATTAGC AGCAGTGGACAGTGCTGTGTATGGAGTCCAAAGAGGAGCCAAAAAGCCCTTGGAAAGAGTATTTCAATTC TTAGAGAAGAGTGATCTGGGCTGTGGGGCAGGTGGTGGCCTCAACAATGCCAATGTGTTCCACCTAGCTG GACTTACCTTCCTCACTAATGCAAATGCAGATGACTCCCAAGAAAATGATGAACCTTGTAAAGAAATTCT CAGGCCAAGAAGAACGCTGCAAAAGAAGATAGAAGAAATAGCTGCTAAATATAAACATTCAGTAGTGAAG AAATGTTGTTACGATGGAGCCTGCGTTAATAATGATGAAACCTGTGAGCAGCGAGCTGCACGGATTAGTT TAGGGCCAAGATGCATCAAAGCTTTCACTGAATGTTGTGTCGTCGCAAGCCAGCTCCGTGCTAATATCTC TCATAAAGACATGCAATTGGGAAGGCTACACATGAAGACCCTGTTACCAGTAAGCAAGCCAGAAATTCGG AGTTATTTTCCAGAAAGCTGGTTGTGGGAAGTTCATCTTGTTCCCAGAAGAAAACAGTTGCAGTTTGCCC TACCTGATTCTCTAACCACCTGGGAAATTCAAGGCATTGGCATTTCAAACACTGGTATATGTGTTGCTGA TACTGTCAAGGCAAAGGTGTTCAAAGATGTCTTCCTGGAAATGAATATACCATATTCTGTTGTACGAGGA GAACAGATCCAATTGAAAGGAACTGTTTACAACTATAGGACTTCTGGGATGCAGTTCTGTGTTAAAATGT CTGCTGTGGAGGGAATCTGCACTTCGGAAAGCCCAGTCATTGATCATCAGGGCACAAAGTCCTCCAAATG TGTGCGCCAGAAAGTAGAGGGCTCCTCCAGTCACTTGGTGACATTCACTGTGCTTCCTCTGGAAATTGGC CTTCACAACATCAATTTTTCACTGGAGACTTGGTTTGGAAAAGAAATCTTAGTAAAAACATTACGAGTGG TGCCAGAAGGTGTCAAAAGGGAAAGCTATTCTGGTGTTACTTTGGATCCTAGGGGTATTTATGGTACCAT TAGCAGACGAAAGGAGTTCCCATACAGGATACCCTTAGATTTGGTCCCCAAAACAGAAATCAAAAGGATT TTGAGTGTAAAAGGACTGCTTGTAGGTGAGATCTTGTCTGCAGTTCTAAGTCAGGAAGGCATCAATATCC TAACCCACCTCCCCAAAGGGAGTGCAGAGGCGGAGCTGATGAGCGTTGTCCCAGTATTCTATGTTTTTCA CTACCTGGAAACAGGAAATCATTGGAACATTTTTCATTCTGACCCATTAATTGAAAAGCAGAAACTGAAG AAAAAATTAAAAGAAGGGATGTTGAGCATTATGTCCTACAGAAATGCTGACTACTCTTACAGTGTGTGGA AGGGTGGAAGTGCTAGCACTTGGTTAACAGCTTTTGCTTTAAGAGTACTTGGACAAGTAAATAAATACGT AGAGCAGAACCAAAATTCAATTTGTAATTCTTTATTGTGGCTAGTTGAGAATTATCAATTAGATAATGGA TCTTTCAAGGAAAATTCACAGTATCAACCAATAAAATTACAGGGTACCTTGCCTGTTGAAGCCCGAGAGA ACAGCTTATATCTTACAGCCTTTACTGTGATTGGAATTAGAAAGGCTTTCGATATATGCCCCCTGGTGAA AATCGACACAGCTCTAATTAAAGCTGACAACTTTCTGCTTGAAAATACACTGCCAGCCCAGAGCACCTTT ACATTGGCCATTTCTGCGTATGCTCTTTCCCTGGGAGATAAAACTCACCCACAGTTTCGTTCAATTGTTT CAGCTTTGAAGAGAGAAGCTTTGGTTAAAGGTAATCCACCCATTTATCGTTTTTGGAAAGACAATCTTCA GCATAAAGACAGCTCTGTACCTAACACTGGTACGGCACGTATGGTAGAAACAACTGCCTATGCTTTACTC ACCAGTCTGAACTTGAAAGATATAAATTATGTTAACCCAGTCATCAAATGGCTATCAGAAGAGCAGAGGT ATGGAGGTGGCTTTTATTCAACCCAGGACACCATCAATGCCATTGAGGGCCTGACGGAATATTCACTCCT GGTTAAACAACTCCGCTTGAGTATGGACATCGATGTTTCTTACAAGCATAAAGGTGCCTTACATAATTAT AAAATGACAGACAAGAATTTCCTTGGGAGGCCAGTAGAGGTGCTTCTCAATGATGACCTCATTGTCAGTA CAGGATTTGGCAGTGGCTTGGCTACAGTACATGTAACAACTGTAGTTCACAAAACCAGTACCTCTGAGGA AGTTTGCAGCTTTTATTTGAAAATCGATACTCAGGATATTGAAGCATCCCACTACAGAGGCTACGGAAAC TCTGATTACAAACGCATAGTAGCATGTGCCAGCTACAAGCCCAGCAGGGAAGAATCATCATCTGGATCCT CTCATGCGGTGATGGACATCTCCTTGCCTACTGGAATCAGTGCAAATGAAGAAGACTTAAAAGCCCTTGT GGAAGGGGTGGATCAACTATTCACTGATTACCAAATCAAAGATGGACATGTTATTCTGCAACTGAATTCG ATTCCCTCCAGTGATTTCCTTTGTGTACGATTCCGGATATTTGAACTCTTTGAAGTTGGGTTTCTCAGTC CTGCCACTTTCACAGTTTACGAATACCACAGACCAGATAAACAGTGTACCATGTTTTATAGCACTTCCAA TATCAAAATTCAGAAAGTCTGTGAAGGAGCCGCGTGCAAGTGTGTAGAAGCTGATTGTGGGCAAATGCAG GAAGAATTGGATCTGACAATCTCTGCAGAGACAAGAAAACAAACAGCATGTAAACCAGAGATTGCATATG CTTATAAAGTTAGCATCACATCCATCACTGTAGAAAATGTTTTTGTCAAGTACAAGGCAACCCTTCTGGA TATCTACAAAACTGGGGAAGCTGTTGCTGAGAAAGACTCTGAGATTACCTTCATTAAAAAGGTAACCTGT ACTAACGCTGAGCTGGTAAAAGGAAGACAGTACTTAATTATGGGTAAAGAAGCCCTCCAGATAAAATACA ATTTCAGTTTCAGGTACATCTACCCTTTAGATTCCTTGACCTGGATTGAATACTGGCCTAGAGACACAAC ATGTTCATCGTGTCAAGCATTTTTAGCTAATTTAGATGAATTTGCCGAAGATATCTTTTTAAATGGATGC TAAAATTCCTGAAGTTCAGCTGCATACAGTTTGCACTTATGGACTCCTGTTGTTGAAGTTCGTTTTTTTG TTTTCTTCTTTTTTTAAACATTCATAGCTGGTCTTATTTGTAAAGCTCACTTTACTTAGAATTAGTGGCA CTTGCTTTTATTAGAGAATGATTTCAAATGCTGTAACTTTCTGAAATAACATGGCCTTGGAGGGCATGAA GACAGATACTCCTCCAAGGTTATTGGACACCGGAAACAATAAATTGGAACACCTCCTCAAACCTACCACT CAGGAATGTTTGCTGGGGCCGAAAGAACAGTCCATTGAAAGGGAGTATTACAAAAACATGGCCTTTGCTT GAAAGAAAATACCAAGGAACAGGAAACTGATCATTAAAGCCTGAGTTTGCTTTC (SEQ ID NO: 6696) gi|189944|gb|L05144.1|HUMPHOCAR Homo sapiens (clone lamda-hPEC-3) phosphoenolpyruvate carboxykinase (PCK1) mRNA, complete cds TGGGAACACAAACTTGCTGGCGGGAAGAGCCCGGAAAGAAACCTGTGGATCTCCCTTCGAGATCATCCAA AGAGAAGAAAGGTGACCTCACATTCGTGCCCCTTAGCAGCACTCTGCAGAAATGCCTCCTCAGCTGCAAA ACGGCCTGAACCTCTCGGCCAAAGTTGTCCAGGGAAGCCTGGACAGCCTGCCCCAGGCAGTGAGGGAGTT TCTCGAGAATAACGCTGAGCTGTGTCAGCCTGATCACATCCACATCTGTGACGGCTCTGAGGAGGAGAAT GGGCGGCTTCTGGGCCAGATGGAGGAAGAGGGCATCCTCAGGCGGCTGAAGAAGTATGACAACTGCTGGT TGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGACGGTTATCGTCACCCAAGAGCAAAG AGACACAGTGCCCATCCCCAAAACAGGCCTCAGCCAGCTCGGTCGCTGGATGTCAGAGGAGGATTTTGAG AAAGCGTTCAATGCCAGGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCCATTCAGCATGG GGCCGCTGGGCTCACCTCTGTCGAAGATCGGCATCGAGCTGACGGATTCGCCCTACGTGGTGGCCAGCAT GCGGATCATGACGCGGATGGGCACGCCCGTCCTGGAAGCACTGGGCGATGGGGAGTTTGTCAAATGCCTC CATTCTGTGGGGTGCCCTCTGCCTTTACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGA CGCTCATCGCCCACCTGCCTGACCGCAGAGAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACTCGCT GCTCGGGAAGAAGTGCTTTGCTCTCAGGATGGCCAGCCGGCTGGCAGAGGAGGAAGGGTGGCTGGCAGAG CACATGCTGATTCTGGGTATAACCAACCCTGAGGGTGAGAAGAAGTACCTGGCGGCCGCATTTCCCAGCG CCTGCGGGAAGACCAACCTGGCCATGATGAACCCCAGCCTCCCCGGGTGGAAGGTTGAGTGCGTCGGGGA TGACATTGCCTGGATGAAGTTTGACGCACAAGGTCATTTAAGGGCCATCAACCCAGAAAATGGCTTTTTC GGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATCAAGACCATCCAGAAGAACACAATCT TTACCAATGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAGGCATTGATGAGCCGCTAGCTTCAGG CGTCACCATCACGTCCTGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTGCCCACCCCAAC TCGAGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTCTCCGGAAGGTGTTC CCATTGAAGGCATTATCTTTGGAGGCCGTAGACCTGCTGGTGTCCCTCTAGTCTATGAAGCTCTCAGCTG GCAACATGGAGTCTTTGTGGGGGCGGCCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAA ATCATCATGCATGACCCCTTTGCCATGCGGCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCCCACT GGCTTAGCATGGCCCAGCACCCAGCAGCCAAACTGCCCAAGATCTTCCATGTCAACTGGTTCCGGAAGGA CAAGGAAGGCAAATTCCTCTGGCCAGGCTTTGGAGAGAACTCCAGGGTGCTGGAGTGGATGTTCAACCGG ATCGATGGAAAAGCCAGCACCAACGTCACGCCCATAGGCTACATCCCCAAGGAGGATGCCCTGAACCTGA AAGGCCTGGGGCACATCAACATGATGGAGCTTTTCAGCATCTCCAAGGAATTCTGGGACAAGGAGGTGGA AGACATCGAGAAGTATCTGGTGGATCAAGTCAATGCCGACCTCCCCTGTGAAATCGAGAGAGAGATCCTT GCCTTGAAGCAAAGAATAAGCCAGATGTAATCAGGGCCTGAGAATAAGCCAGATGTAATCAGGGCCTGAG TGCTTTACCTTTAAAATCATTAAATTAAAATCCATAAGGTGCAGTAGGAGCAAGAGAGGGCAAGTGTTCC CAAATTGACGCCACCTAATAATCATCACCACACCGGGAGCAGATCTGAAGGCACACTTTGATTTTTTTAA GGATAAGAACCACAGAACACTGGGTAGTAGCTAATGAAATTGAGAAGGGAAATCTTAGCATGCCTCCAAA AATTCACATCCAATGCATACTTTGTTCAAATTTAAGGTTACTCAGGCATTGATCTTTTCAGTGTTTTTTC ACTTAGCTATGTGGATTAGCTAGAATGCACACCAAAAAGATACTTGAGCTGTATATATATATGTGTGTGT GTGTGTGTGTGTGTGTGTGTGTGCATGTATGTGCACATGTGTCTGTGTGATATTTGGTATGTGTATTTGT ATGTACTGTTATTCAAAATATATTTAATACCTTTGGAAAATCTTGGGCAAGATGACCTACTAGTTTTCCT TGAAAAAAAGTTGCTTTGTTATTAATATTGTGCTTAAATTATTTTTATACACCATTGTTCCTTACCTTTA CATAATTGCAATATTTCCCCCTTACTACTTCTTGGAAAAAAATTAGAAAATGAAGTTTATAGAAAAG (SEQ ID NO: 6697) gi|6679892|ref|NM_008061.1| Mus musculus glucose-6-phosphatase, catalytic (G6pc), mRNA AGCAGAGGGATCGGGGCCAACCGGGCTTGGACTCACTGCACGGGCTCTGCTGGCAGCTTCCTGAGGTACC AAGGGAGGAAGGATGGAGGAAGGAATGAACATTCTCCATGACTTTGGGATCCAGTCGACTCGCTATCTCC AAGTGAATTACCAAGACTCCCAGGACTGGTTCATCCTTGTGTCTGTGATTGCTGACCTGAGGAACGCCTT CTATGTCCTCTTTCCCATCTGGTTCCATCTTAAAGAGACTGTGGGCATCAATCTCCTCTGGGTGGCAGTG GTCGGAGACTGGTTCAACCTCGTCTTCAAGTGGATTCTGTTTGGACAACGCCCGTATTGGTGGGTCCTGG ACACCGACTACTACAGCAACAGCTCCGTGCCTATAATAAAGCAGTTCCCTGTCACCTGTGAGACCGGACC AGGAAGTCCCTCTGGCCATGCCATGGGCGCAGCAGGTGTATACTATGTTATGGTCACTTCTACTCTTGCT ATCTTTCGAGGAAAGAAAAAGCCAACGTATGGATTCCGGTGTTTGAACGTCATCTTGTGGTTGGGATTCT GGGCTGTGCAGCTGAACGTCTGTCTGTCCCGGATCTACCTTGCTGCTCACTTTCCCCACCAGGTCGTGGC TGGAGTCTTGTCAGGCATTGCTGTGGCTGAAACTTTCAGCCACATCCGGGGCATCTACAATGCCAGCCTC CGGAAGTATTGTCTCATCACCATCTTCTTGTTTGGTTTCGCGCTTGGATTCTACCTGCTACTAAAAGGGC TAGGGGTGGACCTCCTGTGGACTTTGGAGAAAGCCAAGAGATGGTGTGAGCGGCCAGAATGGGTCCACCT TGACACTACACCCTTTGCCAGCCTCTTCAAAAACCTGGGAACCCTCTTGGGGTTGGGGCTGGCCCTCAAC TCCAGCATGTACCGGAAGAGCTGCAAGGGAGAACTCAGCAAGTCGTTCCCATTCCGCTTCGCCTGCATTG TGGCTTCCTTGGTCCTCCTGCATCTCTTTGACTCTCTGAAGCCCCCATCCCAGGTTGAGTTGATCTTCTA CATCTTGTCTTTCTGCAAGAGCGCAACAGTTCCCTTTGCATCTGTCAGTCTTATCCCATACTGCCTAGCC CGGATCCTGGGACAGACACACAAGAAGTCTTTGTAAGGCATGCAGAGTCTTTGGTATTTAAAGTCAACCG CCATGCAAAGGACTAGGAACAACTAAAGCCTCTGAAACCCATTGTGAGGCCAGAGGTGTTGACATCGGCC CTGGTAGCCCTGTCTTTCTTTGCTATCTTAACCAAAAGGTGAATTTTTACAAAGCTTACAGGGCTGTTTG AGGAAAGTGTGAATGCTGGAAACTGAGTCATTCTGGATGGTTCCCTGAAGATTCGCTTACCAGCCTCCTG TCAGATACAGAAGAGCAAGCCCAGGCTAGAGATCCCAACTGAGAATGCTCTTGCGGTGCAGAATCTTCCG GCTGGGAAAAGGAAAAGAGCACCATGCATTTGCCAGGAAGAGAAAGAAGGATCGGGAGGAGGGAGAGTGT TTTATGTATCGAGCAAACCAGATGCAATCTATGTCTAACCGGCTTCAGTTGTGTCTGCGTCTTTAGATAC GACACACTCAATAATAATAATAGACCAACTAGTGTAATGAGTAGCCAGTTAAAGGCGATTAATTCTGCTT CCAGATAGTCTCCACTGTACATAAAAGTCACACTGTGTGCTTGCATTCCTGTATGGTAGTGGTGACTGTC TCTCACACCACCTTCTCTATCACGTCACAGTTTTCTCCTCCTCAGCCTATGTCTGCATTCCCCAGAATTC TCCACTTGTTCCCTGGCCCTGCTGCTGGACCCTGCTGTGTCTGGTAGGCAACTGTTTGTTGGTGCTTTTG TAGGGTTAAGTTAAACTCTGAGATCTTGGGCAAAATGGCAAGGAGACCCAGGATTCTTCTCTCCAAAGGT CACTCCGATGTTATTTTTGATTCCTGGGGCAGAAATATGACTCCTTTCCCTAGCCCAAGCCAGCCAAGAG CTCTCATTCTTAGAAGAAAAGGCAGCCCCTTGGTGCCTGTCCTCCTGCCTCGGCTGATTTGCAGAGTACT TCTTCAAAAAGAAAAAAATGGTAAAGCTATTTATTAAAAATTCTTTGTTTTTTGCTACAAATGATGCATA TATTTTCACCCACACCAAGCACTTTGTTTCTAATATCTTTGATAAGAAAACTACATGTGCAGTATTTTAT TAAAGCAACATTTTATTTA (SEQ ID NO: 6698) gi|7110682|ref|NM_011044.1| Mus musculus phosphoenolpyruvate carboxykinase 1, cytosolic (Pck1), mRNA ACAGTTGGCCTTCCCTCTGGGAACACACCCTCGGTCAACAGGGGAAATCCGGCAAGGCGCTCAGCGATCT CTGATCCAGACCTTCCAAAAGGAAGAAAGGTGGCACCAGAGTTCCTGCCTCTCTCCACACCATTGCAATT ATGCCTCCTCAGCTGCATAACGGTCTGGACTTCTCTGCCAAGGTTATCCAGGGCAGCCTCGACAGCCTGC CCCAGGCAGTGAGGAAGTTCGTGGAAGGCAATGCTCAGCTGTGCCAGCCGGAGTATATCCACATCTGCGA TGGCTCCGAGGAGGAGTACGGGCAGTTGCTGGCCCACATGCAGGAGGAGGGTGTCATCCGCAAGCTGAAG AAATATGACAACTGTTGGCTGGCTCTCACTGACCCTCGAGATGTGGCCAGGATCGAAAGCAAGACAGTCA TCATCACCCAAGAGCAGAGAGACACAGTGCCCATCCCCAAAACTGGCCTCAGCCAGCTGGGCCGCTGGAT GTCGGAAGAGGACTTTGAGAAAGCATTCAACGCCAGGTTCCCAGGGTGCATGAAAGGCCGCACCATGTAT GTCATCCCATTCAGCATGGGGCCACTGGGCTCGCCGCTGGCCAAGATTGGTATTGAACTGACAGACTCGC CCTATGTGGTGGCCAGCATGCGGATCATGACTCGGATGGGCATATCTGTGCTGGAGGCCCTGGGAGATGG GGAGTTCATCAAGTGCCTGCACTCTGTGGGGTGCCCTCTCCCCTTAAAAAAGCCTTTGGTCAACAACTGG GCCTGCAACCCTGAGCTGACCCTGATCGCCCACCTCCCGGACCGCAGAGAGATCATCTCCTTTGGAAGCG GATATGGTGGGAACTCACTACTCGGGAAGAAATGCTTTGCGTTGCGGATCGCCAGCCGTCTGGCTAAGGA GGAAGGGTGGCTGGCGGAGCATATGCTGATCCTGGGCATAACTAACCCCGAAGGCAAGAAGAAATACCTG GCCGCAGCCTTCCCTAGTGCCTGTGGGAAGACTAACTTGGCCATGATGAACCCCAGCCTGCCCGGGTGGA AGGTCGAATGTGTGGGCGATGACATTGCCTGGATGAAGTTTGATGCCCAAGGCAACTTAAGGGCTATCAA CCCAGAAAACGGGTTTTTTGGAGTTGCTCCTGGCACCTCAGTGAAGACAAATCCAAATGCCATTAAAACC ATCCAGAAAAACACCATCTTCACCAACGTGGCCGAGACTAGCGATGGGGGTGTTTACTGGGAAGGCATCG ATGAGCCGCTGGCCCCGGGAGTCACCATCACCTCCTGGAAGAACAAGGAGTGGAGACCGCAGGACGCGGA ACCATGTGCCCATCCCAACTCGAGATTCTGCACCCCTGCCAGCCAGTGCCCCATTATTGACCCTGCCTGG GAATCTCCAGAAGGAGTACCCATTGAGGGTATCATCTTTGGTGGCCGTAGACCTGAAGGTGTCCCCCTTG TCTATGAAGCCCTCAGCTGGCAGCATGGGGTGTTTGTAGGAGCAGCCATGAGATCTGAGGCCACAGCTGC TGCAGAACACAAGGGCAAGATCATCATGCACGACCCCTTTGCCATGCGACCCTTCTTCGGCTACAACTTC GGCAAATACCTGGCCCACTGGCTGAGCATGGCCCACCGCCCAGCAGCCAAGTTGCCCAAGATCTTCCATG TCAACTGGTTCCGGAAGGACAAAGATGGCAAGTTCCTCTGGCCAGGCTTTGGCGAGAACTCCCGGGTGCT GGAGTGGATGTTCGGGCGGATTGAAGGGGAAGACAGCGCCAAGCTCACGCCCATCGGCTACATCCCTAAG GAAAACGCCTTGAACCTGAAAGGCCTGGGGGGCGTCAACGTGGAGGAGCTGTTTGGGATCTCTAAGGAGT TCTGGGAGAAGGAGGTGGAGGAGATCGACAGGTATCTGGAGGACCAGGTCAACACCGACCTCCCTTACGA AATTGAGAGGGAGCTCCGAGCCCTGAAACAGAGAATCAGCCAGATGTAAATCCCAATGGGGGCGTCTCGA GAGTCACCCCTTCCCACTCACAGCATCGCTGAGATCTAGGAGAAAGCCAGCCTGCTCCAGCTTTGAGATA GCGGCACAATCGTGAGTAGATCAGAAAAGCACCTTTTAATAGTCAGTTGAGTAGCACAGAGAACAGGCTA GGGGCAAATAAGATTGGGAGGGGAAATCACCGCATAGTCTCTGAAGTTTGCATTTGACACCAATGGGGGT TTTGGTTCCACTTCAAGGTCACTCAGGAATCCAGTTCTTCACGTTAGCTGTAGCAGTTAGCTAAAATGCA CAGAAAACATACTTGAGCTGTATATATGTGTGTGAACGTGTCTCTGTGTGAGCATGTGTGTGTGTGTGTG TGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTACATGCCTGTCTGTCCCATTGTCCACAGTATATTTAA AACCTTTGGGGAAAAATCTTGGGCAAATTTGTAGCTGTAACTAGAGAGTCATGTTGCTTTGTTGCTAGTA TGTATGTTTAAATTATTTTTATACACCGCCCTTACCTTTCTTTACATAATTGAAATTGGTATCCGGACCA CTTCTTGGGAAAAAAATTACAAAATAAA (SEQ ID NO: 6699)

Example 6 siRNAs Decrease mRNA Levels In Vivo

Male CMV-Luc mice (8-10 weeks old) from Xenogen (Cranbury, N.J.) were administered cholesterol conjugated siRNA (see Table 16).

TABLE 16 Solutions adminstered to mice Group n Injection Mix 1 7 Buffer (PBS [pH 7.4]) 2 8 Cholesterol conjugated siRNA (ALN-3001)

TABLE 17 Test iRNA agents targeting Luciferase siRNA Sequence ALN-1070 5′-GAA CUG UGU GUG AGA GGU CCU-3′ (SEQ ID NO: 6700) 3′-CG CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6701) ALN-1000 5′-GAA CUG UGU GUG AGA GGU CCU-GS-3′ (SEQ ID NO: 6702) 3′-CG CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6703) ALN-3000 5′-GAA CUG UGU GUG AGA GGU CCU-3′ (SEQ ID NO: 6704) 3′-Cs¹Gs¹ CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6705) ALN-3001 5′-GAA CUG UGU GUG AGA GGU CCU-chol.²-3′ (SEQ ID NO: 6706) 3′-Cs¹Gs¹ CUU GAC ACA CAC UCU CCA GGA-5′ (SEQ ID NO: 6707) ¹2′ O-Me group is attached to the nucleotide and the nucleotides have phosphorothioate linkages (indicated by “s”) ²cholesterol is conjugated to the antisense strand via the linker: U-pyrroline carrier-C(O)-(CH2)5-NHC(O)-cholesterol (via cholesterol C-3 hydroxyl).

Animals were injected (tail vein) with a volume of 200-250 μl test solution containing buffer or an siRNA solution. Group 1 received buffer and group 2 received cholesterol conjugated siRNA (ALN-3001) at a dose of 50 mg/kg body weight. Twenty-two hours after injection, animals were sacrificed and livers collected. Organs were snap frozen on dry ice, then pulverized in a mortar and pestle.

For Luciferase mRNA analysis (by the QuantiGene Assay (Genospectra, Inc.; Fremont, Calif.)), approximately 10 mg of tissue powder was resuspended in tissue lysis buffer, and processed according to the manufacturer's protocol. Samples of the lysate were hybridized with probes specific for Luciferase or GAPDH (designed using ProbeDesigner software (Genospectra, Inc., Fremont, Calif.) in triplicate, and processed for luminometric analysis. Values for Luciferase were normalized to GAPDH. Mean values were plotted with error bars corresponding to the standard deviation of the Luciferase measurements.

Results indicated that the level of luciferase RNA in animals injected with cholesterol conjugated siRNA was reduced by about 70% as compared to animals injected with buffer (see FIGS. 6A and 6 b).

In Vitro Activity

HeLa cells expressing luciferase were transfected with each of the siRNAs listed in Table 17. ALN-1000 siRNAs were most effective at decreasing luciferase mRNA levels (˜0.6 nM siRNA decreased mRNA levels to about ˜65% the original expression level, and 1.0 nM siRNA decreased levels to about ˜20% the original expression level); ALN-3001 siRNAs were least effective (˜0.6 nM siRNA had a negligible mRNA levels, and 1.0 nM siRNA decreased levels to about ˜40% the original expression level).

Pharmacokinetics/Biodistribution

Pharmacokinetic analyses were performed in mice and rats. Test siRNA molecules were radioactively labeled with ³³P on the antisense strand by splint ligation. Labeled siRNAs (50 mg/kg) were administered by tail vein injection, and plasma levels of siRNA were measured periodically over 24 hrs by scintillation counting. Cholesterol conjugated siRNA (ALN-3001) was discovered to circulate in mouse plasma for a longer period time than unconjugated siRNA (ALN-3000) (FIG. 7). RNAse protection assays indicated that cholesterol-conjugated siRNA (ALN-3001) was detectable in mouse plasma 12 hours after injection, whereas unconjugated siRNA (ALN-3000) was not detectable in mouse plasma within two hours following injection. Similar results were observed in rats.

Mouse liver was harvested at varying time points (ranging from 0.08-24 hours) following injection with siRNA, and siRNA localized to the liver was quantified. Over the time period tested, the amount of cholesterol-conjugated siRNA (ALN-3001) detected in the liver ranged from 14.3-3.55 percent of the total dose administered to the mouse. The amount of unconjugated siRNA (ALN-3000) detected in the liver was lower, ranging from 3.91-1.75 percent of the total dose administered.

Detection of siRNA in Different Tissues

Various tissues and organs (fat, heart, kidney, liver, and spleen) were harvested from two CMV-Luc mice 22 hours following injection with 50 mg/kg ALN-3001. The antisense strand of the siRNA was detected by RNAse protection assay. The liver contained the greatest concentration of siRNA (˜8-10 μg siRNA/g tissue); the spleen, heart and kidney contained lesser amounts of siRNA (˜2-7 μg siRNA/g tissue); and fat tissue contained the least amount of siRNA (<˜1 μg siRNA/g tissue).

Glucose-6-phosphatase siRNA Detection by RNAse Protection Assay

Balbc mice were injected with U/U, 3′C/U, or 3′C/3′C siRNA (4 mg/kg) targeting glucose-6-phosphatase (G6Pase) (see Table 18). Administration was by hydrodynamic tail vein injection (hd) or non-hydrodynamic tail vein injection (iv), and siRNA was subsequently detected in the liver by RNAse protection assay.

TABLE 18 Test iRNA agents targeting glucose-6-phosphatase siRNA Description U/U No cholesterol; dinucleotide 3′ overhangs on sense and antisense strands 3′C/U dinucleotide 3′ overhangs on sense and antisense strands; cholesterol conjugated to 3′ end of sense strand (mono-conjugate) 3′C/3′C dinucleotide 3′ overhangs on sense and antisense strands; cholesterol conjugated to 3′ end of both sense and antisense strands (bis-conjugate)

Unconjugated siRNA (U/U) delivered by hd was detected by 15 min. post-injection (the earliest determined time-point) and was still detectable in the liver 18 hours post-injection.

Delivery by normal iv administration resulted in the greatest concentration of 3′C/3′C siRNA (the bis-cholesterol-conjugate) in the liver 1 hour post injection (as compared to the mono-cholesterol-conjugate 3′C/3′U siRNA). At 18 hours post injection, 3′C/3′C siRNAs and 3′C/U siRNA were still detectable in the liver with the bis-conjugate at higher levels compared to the mono-conjugate.

While this invention has been particularly shown and described with reference to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the scope of the invention encompassed by the appended claims. 

What is claimed is:
 1. An iRNA agent, comprising: a sense strand sequence having 4-12 asymmetrical 2′-O-alkyl modifications, at least 4 of which occur within the 6 terminal nucleotides of the 5′ end; and an antisense sequence having at least 4 asymmetrical phosphorothioate modifications.
 2. The iRNA agent of claim 1, wherein the sense and antisense sequences of the iRNA agent are on different RNA strands.
 3. The iRNA agent of claim 1, wherein the sense and antisense sequences of the iRNA agent are on the same RNA strand.
 4. The iRNA agent of claim 1, wherein the sense and antisense sequences of the iRNA agent are fully complementary to each other.
 5. The iRNA agent of claim 1, further comprising a cholesterol moiety, wherein the cholesterol moiety is coupled to the sense sequence of the iRNA agent.
 6. The iRNA agent of claim 1, wherein the iRNA agent is at least 21 nucleotides in length, and the duplex region of the iRNA agent is about 19 nucleotides in length.
 7. The iRNA agent of claim 1, the iRNA agent having a duplex region of about 19 nucleotides in length and one or two 3′ overhangs of about 2 nucleotides in length.
 8. The iRNA agent of claim 1, wherein the antisense sequence contains 4-20 asymmetrical phosphorothioate modifications.
 9. The iRNA agent of claim 1, wherein each of the 2′-O-alkyl modifications are 2′-O-methyl modifications.
 10. The iRNA agent of claim 1, wherein the sense strand further comprises at least one asymmetric modification selected from the group consisting of 2′-5′-linkages, L sugars, modified sugars, nucleobase modifications, cation groups, Zwitterionic groups, and conjugate groups.
 11. The iRNA agent of claim 10, wherein the modification is 2′-5′ linkages, and the 2′-5′ linkage is phosphorothioate.
 12. The iRNA agent of claim 10, wherein the modification is L sugars, and the L sugar is L ribose or L-arabinose sugar.
 13. The iRNA agent of claim 10, wherein the modification is modified sugars, and the modified sugar is a locked nucleic acid, hexose nucleic acid or cyclohexane nucleic acid.
 14. The iRNA agent of claim 10, wherein the conjugate group is naproxen, biotin, ibuprofen, folic acid, peptides or carbohydrates.
 15. The iRNA agent of claim 1, wherein the antisense strand further comprises at least one asymmetric modification selected from the group consisting of 2′-5′-linkages, L sugars, modified sugars, nucleobase modifications, cation groups, Zwitterionic groups, and conjugate groups.
 16. The iRNA agent of claim 15, wherein the modification is 2′-5′ linkages, and the 2′-5′ linkage is phosphorothioate.
 17. The iRNA agent of claim 15, wherein the modification is L sugars, and the L sugar is L ribose or L-arabinose sugar.
 18. The iRNA agent of claim 15, wherein the modification is modified sugars, and the modified sugar is a locked nucleic acid, hexose nucleic acid or cyclohexane nucleic acid.
 19. The iRNA agent of claim 1, wherein the sense strand has 4 asymmetrical 2′-O-alkyl modifications within the 6 terminal nucleotides of the 5′ end, and at least one 2′-O-alkyl modification in another portion of the sense strand.
 20. The iRNA agent of claim 15, wherein antisense strand does not contain any 2′-O-alkyl modifications, and the sense strand does not contain any phosphorothioate modifications. 